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MOLÉCULAS DE FUSÃO E FATORES TRANSCRICIONAIS
EM MACRÓFAGOS E CÉLULAS MUSCULARES
ESQUELÉTICAS DE RATOS: EFEITO DA
DESNUTRIÇÃO NEONATAL
JULIANA FÉLIX DE MELO
RECIFE/PE
2012
Thèse présentée
par
Juliana Félix de Melo
MOLÉCULES DE FUSION ET FACTEURS DE
TRANSCRIPTION DANS LES MACROPHAGES ET
CELLULES MUSCULAIRES SQUELETTIQUES DE
RATS: L’EFFET DE LA DÉNUTRITION NÉONATALE
Pour l’obtention du grade de Docteur de
l’Université de Technologie de Compiègne
(UTC) et de l’Université Fédérale de
Pernambuco (UFPE)
Thèse en co-tutelle dirigée par:
Marie-Danielle Nagel, UMR 6600, UTC – France
Muriel Vayssade, UMR 6600, UTC – France
Célia Maria Machado Barbosa de Castro, Departamento de Medicina Tropical,
UFPE - Brésil
RECIFE/PE
2012
Tese apresentada
por
Juliana Félix de Melo
MOLÉCULAS DE FUSÃO E FATORES TRANSCRICIONAIS
EM MACRÓFAGOS E CÉLULAS MUSCULARES
ESQUELÉTICAS DE RATOS: EFEITO DA
DESNUTRIÇÃO NEONATAL
Para obtenção do título de Doutor do
Programa de Pós-Graduação em Medicina
Tropical do Centro de Ciências da Saúde da
Universidade Federal de Pernambuco e da
Escola de Doutorado da Universidade de
Tecnologia de Compiègne
Tese co-tutela orientada por:
Célia Maria Machado Barbosa de Castro, Departamento de Medicina Tropical,
UFPE – Brasil
Marie-Danielle Nagel, UMR 6600, UTC – França
Muriel Vayssade, UMR 6600, UTC – França
RECIFE/PE
2012
Melo, Juliana Félix de
Moléculas de fusão e fatores transcricionais em macrófagos e células musculares esqueléticas de ratos: efeito da desnutrição neonatal / Juliana Félix de Melo. – Recife: O Autor, 2012.
100 folhas: il., fig. ; 30 cm.
Orientador: Célia Maria Machado Barbosa de Castro. Tese (doutorado) – Universidade Federal de Pernambuco. CCS. Medicina Tropical, 2012.
Inclui bibliografia e anexos.
1. Desnutrição. 2. Fusão celular. 3. Macrófagos
alveolares. 4. Músculo esquelético. 5. Mioblastos. I. Castro,
Célia Maria Machado Barbosa de. II. Título.
UFPE 616.079 CDD (20.ed.) CS2012-058
UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINA TROPICAL
REITOR
Anísio Brasileiro de Freitas Dourado
PRÓ-REITOR PARA ASSUNTOS DE PESQUISA E PÓS-GRADUAÇÃO
Francisco de Sousa Ramos
DIRETOR DO CENTRO DE CIÊNCIAS DA SAÚDE
José Thadeu Pinheiro
COORDENADORA DO PROGRAMA DE PÓS-GRADUAÇÃO
EM MEDICINA TROPICAL
Maria Rosângela Cunha Duarte Coêlho
VICE-COORDENADORA DO PROGRAMA DE PÓS-GRADUAÇÃO
EM MEDICINA TROPICAL
Valdênia Maria Oliveira de Souza
CORPO DOCENTE
Ana Lúcia Coutinho Domingues
Célia Maria Machado Barbosa de Castro
Edmundo Pessoa de Almeida Lopes Neto
Fábio André Brayner dos Santos
Heloísa Ramos Lacerda de Melo
Maria Amélia Vieira Maciel
Maria de Fátima Pessoa Militão de Albuquerque
Maria do Amparo Andrade
Maria Rosângela Cunha Duarte Coêlho
Marli Tenório Cordeiro
Ricardo Arraes de Alencar Ximenes
Valdênia Maria Oliveira de Souza
Vera Magalhães da Silveira
Vlaúdia Maria Assis Costa
DEDICATÓRIA
A DEUS, cuja presença traz-me sempre a
segurança necessária para enfrentar meu
caminho e seguir.
Aos meus pais, Maurílio Araújo Campelo de
Melo e Nadja Maria Félix dos Santos, por todo
o apoio e incentivo a mim dedicados.
À memória de meus avós: Adauto Campelo de
Melo e Onadir Araújo Campelo de Melo (Didi),
que apreciavam os estudos de forma
indescritível.
AGRADECIMENTOS
À minha orientadora, Célia Maria Machado Barbosa de Castro: Muito obrigada por
todo incentivo e entusiasmo, pelas críticas sempre tão pertinentes e pela confiança sempre!!
Tenho orgulho de ser sua orientanda durante quase 10 anos!! Foi com você que aprendi a ter
ânimo e coragem para seguir minha trajetória na vida acadêmica!!
À minha orientadora, Marie Danielle Nagel: desde a minha chegada na UTC esteve
sempre presente e suas idéias foram fundamentais para o bom planejamento da pesquisa.
Merci pour tout Madame Nagel!! Ma profonde gratitude pour votre patience et vos précieux
conseils.
À pesquisadora Muriel Vayssade, pela disponibilidade e contribuição dada a esta tese.
Às professoras Maria Elizabeth Cavalcante Chaves e Carol Virginia Gois Leandro,
pelo inestimável apoio para realização dos experimentos e finalização dos artigos.
Às amigas Thacianna Costa, Danielle Moura, Adriana Andrade e Natália Cavalcanti,
pela importante contribuição que deram a este trabalho.
À Tamara De’Carli, pela amizade e apoio nos experimentos realizados no Centro de
Pesquisas Aggeu Magalhães.
À Christian Robson, do Centro de Pesquisas Aggeu Magalhães, pelos conselhos
pertinentes a esta pesquisa.
Ao Sr. Walter Leite Galdino e Alice Lourenço, pelo carinho dedicado e colaboração a
todos os alunos da Pós-Graduação em Medicina Tropical.
Ao veterinário Dr. Edeones França, pelos ensinamentos, amizade e apoio no manuseio
dos animais.
Ao Sr. Paulino Ventura, in memorian, pelo apoio com os animais, e principalmente
pelo carinho e amizade que levarei comigo pra sempre. Obrigada por tudo Sr. Paulino!!!
À Roberta Leite, Karla Mônica e Prof. Raul Manhães, pelo apoio e esclarecimentos
prestados referentes à co-tutela.
Gostaria de agradecer a todos os funcionários, alunos e professores que conviveram
comigo no Laboratório de Imunopatologia Keizo Asami (LIKA), durante todo o período
enquanto estive pesquisando no Setor de Microbiologia, e em especial, àqueles que dedicaram
seu tempo, conhecimentos e entusiasmo a este trabalho: Alice Bezerra, André Galvão, Flávia
Regina, Thales Henrique, Thacianna Costa, Natália Morais, Maria do Amparo, Maria de
Fátima, Rafael Padilha, Gabriela Ayres, José Lamartine, Rosângela Coêlho, José Luiz, Fábio
Brayner, Liliane Melo, Rodrigo Fragoso, Érika Michelle, Rebecca Peixoto, Suzan Diniz,
Renata Raele, Guilherme Firmino, Guilherme Magalhães, Danielly Cantarelli, Ketlin Ribas,
Thays Almeida, Ana Valéria, Maiara Severo, Kedma Magalhães, Wylla Tatiana, Simone
Fraga, Felipe Viegas, Maria Helena, Edson Silva, Paulo Freitas, Otaviano Costa, Ilma Santos,
Roberto Afonso, Marcela Silvestre, Conceição Gomes, Monique Ferraz, Rosangela Rosendo,
Larissa Avelar, Mariana Arruda, Bruno Galvão, Carlos Weber, Américo Thomaz, António
Tony, Karla Melo, Bruno Sampaio, Solange Porto.
Aos colegas do doutorado, pelo companheirismo em todos os momentos.
À equipe da Université de Technologie de Compiègne: só tenho a agradecer por todo o
conhecimento e o crescimento que vocês me passaram!! Meus sinceros agradecimentos à
Nijez Aloulou, Pascale Vigneron, Jean-Luc Duval, Muriel Vayssade, Oumou Goundiam,
Marie-Charlotte Bernier, Véronique Belley-Vézina, Murielle Dufresne-Freville, Vanessa
Bleicher, Marie-José Fleury, Alain Le Verger, Odile Leclerc, Chantal Pérot, Catherine
Lacourt, Catherine Marque e Francis Canon.
Aos professores da disciplina de Microbiologia e Imunologia da UFPE, pelo apoio e
incentivo.
Às amigas Kelly Feitosa, Fernanda César, Renata Fabiana, Alexandra Silvestre,
Amanda Mesquita, Ana Luiza e Ivana Porto, pelo incentivo e convivência desde a época da
graduação.
Aos meus familiares (tios, primos) por todo o carinho, em especial, aos meus avós
João Félix e Maria do Carmo (Carminha) sempre muito interessados em meus estudos.
À Antonio Ricardo e à Maria Augusta, pelo carinho e apoio em todos estes anos.
Aos meus pais, Maurílio Melo e Nadja dos Santos, pela compreensão, amor e
incentivo.
Às minhas irmãs: Marina, Marcela e Alice, pelo amor, amizade e carinho.
Ao Programa de Pós-Graduação em Medicina Tropical, ao Programa de Pós-
Graduação em Nutrição e à Pró-Reitoria de Pesquisa e Pós-Graduação da Universidade
Federal de Pernambuco, pela oportunidade de levar adiante minhas pesquisas através dos
auxílios financeiros prestados.
Ao Núcleo de Telessaúde da Universidade Federal de Pernambuco (NUTES/UFPE),
pelo apoio para a realização da videoconferência.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e ao
CAPESCOFECUB do Ministério da Educação e Cultura, pela concessão da bolsa de estudo.
A todos aqueles que, de forma direta ou indireta, contribuíram generosamente para a
concretização desta tese.
“Penso que só há um caminho para a ciência ou para a filosofia: encontrar um
problema, ver a sua beleza e apaixonar-se por ele; casar e viver feliz com ele até que a
morte vos separe – a não ser que encontrem um outro problema ainda mais fascinante,
ou, evidentemente, a não ser que obtenham uma solução. Mas, mesmo que obtenham
uma solução, poderão então descobrir, para vosso deleite, a existência de toda uma
família de problemas-filhos, encantadores ainda que talvez difíceis, para cujo bem-
estar poderão trabalhar, com um sentido, até ao fim dos vossos dias.”
(Karl Popper)
“A mente que se abre a uma nova idéia jamais voltará ao seu tamanho original.”
(Albert Einstein)
RESUMO
Nesta tese avaliamos os efeitos tardios da desnutrição neonatal sobre a expressão/produção de
moléculas de fusão e fatores transcricionais em macrófagos alveolares e células musculares
esqueléticas. Foram utilizados 36 ratos, machos, Wistar, amamentados por mães que
receberam dieta durante a lactação contendo 17% de caseína, grupo nutrido (N) ou 8% de
caseína, grupo desnutrido (D). Após o desmame, os animais foram recuperados com dieta
Labina ou Teklad Global, até 42 dias (n=12), 60 dias (n=12) e 90 dias de vida (n=12). Metade
destes animais (n=18) foi submetida a um procedimento cirúrgico de traqueostomia
objetivando a retirada de lavado broncoalveolar e posterior cultura dos macrófagos alveolares
por 4 dias. Da outra metade (n=18), foram retirados todos os músculos de ambas as patas dos
animais e realizada a cultura das células musculares esqueléticas durante 10 dias. Deste modo,
os resultados geraram dois artigos originais. O primeiro intitulado “Long-term effects of a
neonatal low-protein diet in rats on the number of macrophages in culture and the
expression/production of fusion proteins” permitiu observar que a desnutrição durante a
lactação alterou o número de macrófagos em cultura e a produção de proteínas de fusão em
ratos jovens e adultos, mas não modificou a expressão das moléculas de adesão caderinas. O
segundo intitulado “Effect of a neonatal low-protein diet on the morphology of myotubes in
culture and the expression of key proteins that regulate myogenesis in young and adult rats”
demonstrou que a desnutrição neonatal não modificou a expressão de proteínas-chaves do
processo miogênico, mas alterou a morfologia e reduziu o número dos miotubos em cultura de
animais com 60 dias de vida. Em conclusão, a desnutrição neonatal causou sequelas no
organismo jovem e adulto, mesmo após a reposição nutricional. Estas alterações foram
evidenciadas no desenvolvimento de macrófagos alveolares em cultura e na miogênese.
Palavras chaves: Desnutrição; “Programming”; Fusão de macrófago; Músculo esquelético;
Células satélites; Fusão de mioblasto.
ABSTRACT
In this thesis, we evaluated the late effects of neonatal undernutrition on the
expression/production of fusion molecules and transcriptional factors in alveolar macrophages
and skeletal muscle cells. Thirty-six male Wistar rats were suckled by mothers fed diets
containing 17% casein, control group (C) or 8% casein, undernourished group (UN) during
lactation. After weaning, all animals received a normoproteic diet (Labina or Teklad Global),
at 42 days (n=12), 60 days (n=12) and 90 days (n=12). Half of these animals (n=18) were
submitted to a tracheostomy for the removal of bronchoalveolar lavage and subsequent culture
of alveolar macrophages for 4 days. In the other half (n = 18), all muscles of both legs were
removed and the skeletal muscle cells cultured for 10 days. This resulted in two original
articles. The first of these, entitled “Long-term effects of a neonatal low-protein diet in rats on
the number of macrophages in culture and the expression/production of fusion proteins”,
allowed us to observe that undernutrition during lactation altered the number of macrophages
in culture and the production of fusion proteins in young and adult rats, but did not modify the
expression of cadherin adhesion molecules. The second article, entitled “Effect of a neonatal
low-protein diet on the morphology of myotubes in culture and the expression of key proteins
that regulate myogenesis in young and adult rats”, demonstrated that neonatal undernutrition
did not modify the expression of key proteins of the myogenic process but altered the
morphology and reduced the number of myotubes in culture from 60-day-old rats. In
conclusion, neonatal undernutrition caused sequelae in young and adult organisms, even after
nutritional recovery. These changes were evidenced in the development of alveolar
macrophages in culture and myogenesis.
Keywords: Undernutrition; Programming; Macrophage fusion; Skeletal muscle; Satellite
cells; Myoblast fusion.
RÉSUMÉ
Cette thèse en co-tutelle a été effectuée à l’Université de Technologie de Compiègne
(UTC - France) et à l’Université Fédérale de Pernambuco (UFPE - Brésil). En France, les
activités ont été réalisées dans l’unité de Biomécanique-Bioingénierie (CNRS UMR 6600) de
l’UTC, et au Brésil, dans le Laboratoire de Physiologie de la Nutrition Naíde Teodósio
(LAFINNT) du Département de Nutrition de l’UFPE, dans le Laboratoire
d’Immunopathologie Keizo Asami (LIKA-UFPE) et au sein du Centre de Recherche Aggeu
Magalhães (Fiocruz PE).
Une déficience nutritionnelle pendant la période critique du développement des systèmes
organiques, comme par exemple les systèmes immunitaire et musculaire squelettique, peut
générer des conséquences délétères au niveau structural et fonctionnel de ces systèmes chez
l’adulte (OZANNE et HALES, 2002 ; LUCAS, 2005).
Ainsi, il a été mis en évidence qu’une dénutrition protéino-calorique provoquait une
réduction de l’immunité à médiation cellulaire, de la fonction phagocytaire, de celle du
système du complément, de la production d'anticorps, de la libération de cytokines et de
l'expression des molécules d'adhésion cellulaire (CHANDRA, 2002 ; LANDGRAF et al.,
2005). Dans cette situation, les mécanismes de défense pulmonaire sont touchés avec une
diminution du nombre de macrophages alvéolaires (MA). De plus, plusieurs fonctions des
macrophages sont affectées par la dénutrition néonatale (MELO et al., 2008; FERREIRA E
SILVA et al., 2009). Pour combattre l’infection, les macrophages peuvent fusionner en
réponse à des agents pathogènes et corps étrangers, donnant lieu à des ostéoclastes (os) ou des
cellules géantes multinucléées (CGM) (dans divers tissus). Des études montrent que la E-
cadhérine contribue à la formation des CGM par l’intermédiaire de la cytokine IL-4
(MORENO et al., 2007; HELMING et GORDON, 2009). En plus de l’IL-4, l’interféron γ
(IFN-γ) peut également induire la formation des CGM (HELMING et GORDON, 2008,
2009).
D’autre part, une dénutrition précoce est capable de diminuer la capacité de différenciation
des cellules musculaires et de réduire le nombre de fibres présentes dans le muscle (WILSON
et al., 1988; BAYOL et al., 2004). Les rats dont les mères ont été dénutries par un régime à
base de caséine 8% présentent une réduction du nombre de fibres musculaires et une
diminution de la formation de myotubes secondaires (WILSON et al., 1988). De plus, Bayol
et al. (2004) ont montré que la dénutrition pendant la gestation réduisait la cellularité dans le
muscle après 3 semaines de restauration nutritionnelle. Cependant, peu d’études ont été
consacrées à l’effet d’une dénutrition infligée pendant la période de lactation, sur les
propriétés des cellules musculaires de l’adulte.
Certaines molécules d'adhésion cellulaire telles que les cadhérines et les intégrines sont
importantes pour la première phase de fusion des cellules musculaires squelettiques
(myoblaste x myoblaste), conduisant à la néoformation de myotubes. Horsley et al. (2003) ont
démontré que ces myotubes primaires sécrétaient de l’IL-4, qui interagit avec son récepteur
(IL-4Rα) présent sur les myoblastes pour promouvoir leur fusion avec les myotubes
préexistants (myoblaste x myotube), augmentant ainsi la taille des myotubes. Les facteurs de
transcription Pax7 et myogénine sont importants pour le développement musculaire. Pax7 est
exprimé dans les cellules satellites quiescentes et en phase de prolifération (FOSCHINI et al.,
2004). La myogénine est exprimée dans tous les myoblastes depuis le début de leur
différenciation et son expression est maintenue lors de la fusion des cellules, marquant aussi la
fin de la prolifération des myoblastes (TE PAS et al., 1999).
Le but de notre étude a été de déterminer quelle influence pouvait, chez des rats jeunes et
adultes, avoir l’application d’un régime hypoprotéiné au cours de la période de lactation, sur
l’expression/production des molécules de fusion dans les macrophages alvéolaires et sur celle
de protéines-clés impliquées dans le développement et la différenciation des cellules
musculaires squelettiques.
Nous avons utilisé 36 rats, mâles, Wistar, allaités par des mères ayant été nourries avec
un régime contenant 17% de caséine dans le groupe contrôle (C) ou 8% de caséine dans le
groupe dénutri (D), pendant la période de l’allaitement. Après le sevrage, les animaux ont
été restaurés avec l’alimentation Labina ou de Teklad Global, jusqu’à 42 jours (n=12), 60
jours (n=12) et 90 jours (n=12). La moitié de ces animaux (n=18) a subi une trachéotomie
dans le but de pratiquer un lavage bronchoalvéolaire pour ensuite cultiver des macrophages
alvéolaires pendant 4 jours. L’autre moitié (n=18), a servi à prélever tous les muscles des
deux pattes postérieures de l’animal et à maintenir en culture les cellules musculaires
squelettiques pendant 10 jours. Le poids des rats a été enregistré tous les cinq jours pendant
les 21 premiers jours après la naissance, puis une fois par semaine, afin de surveiller le gain
de poids pendant la phase de récupération nutritionnelle. Le nombre total de cellules a été
évalué indirectement par la libération de l’enzyme lactate déshydrogénase (LDH) après lyse
cellulaire provoquée. Nous avons examiné l’expression de Pan-cadhérine (dans les
macrophages) et de Pan-cadhérine, β1-intégrine, IL-4Rα, Pax7 et myogénine (dans les
cellules musculaires) par Western Blot. Les productions d’IL-4 (dans les macrophages et les
cellules musculaires) et IFN-γ (dans les macrophages) ont été mesurées par ELISA.
Les résultats de cette thèse sont rapportés dans (02) articles originaux. Le premier
(développé à l’UFPE) intitulé “Long-term effects of a neonatal low-protein diet in rats on
the number of macrophages in culture and the expression/production of fusion proteins”
indique que la dénutrition pendant la lactation affecte le nombre de macrophages dans les
cultures cellulaires et la production de protéines de fusion chez les rats jeunes et adultes,
mais ne modifie pas l’expression des cadhérines. Le deuxième (développé à l’UTC), intitulé
“Effect of a neonatal low-protein diet on the morphology of myotubes in culture and the
expression of key proteins that regulate myogenesis in young and adult rats” montre que la
dénutrition néonatale ne modifie pas l’expression de protéines clés du processus
myogénique mais change la morphologie et réduit le nombre de myotubes dans les cultures
obtenues à partir des animaux âgés de 60 jours. Des analyses complémentaires portant sur
les paramètres de contraction des myotubes mesurés chez les animaux dénutris et leurs
témoins ont été réalisées dans le cadre du projet de thèse de Simone Fraga.
En conclusion, la dénutrition néonatale provoque des séquelles chez les organismes
jeunes et adultes, même après restauration nutritionnelle. Ces changements observés à partir
de cultures cellulaires sont évidents dans le développement des macrophages alvéolaires et
dans la myogenèse.
Comme perspectives de ce travail, il peut être envisagé d’évaluer les repercussions de
la dénutrition induite dans la période pré-natale sur la fusion des macrophages et des cellules
musculaires squelettiques lorsque les animaux sont soumis à une activité physique, mais aussi
chez des animaux rendus septiques (durant l’infection on peut noter une perte de muscle
généralisée et l’activité physique pourrait moduler ce processus); de réaliser des co-cultures
de macrophages alvéolaires et de myotubes nouvellement formés et vérifier l’effet de la
dénutrition précoce sur l’index de fusion de ces cellules in vitro. En effet, selon Chargé
(2003), la voie de signalisation qui active la fusion des cellules musculaires active aussi celle
des macrophages, ce qui suggère la possibilité de fusion illégitime entre macrophages et
myotubes.
Mots-clé: Dénutrition; “Programming”; Fusion de macrophage; Muscle squelettique; Cellules
satellites; Fusion de myoblastes.
RÉFÉRENCES
BAYOL, S. et al. The influence of undernutrition during gestation on skeletal muscle
cellularity and on the expression of genes that control muscle growth. Br. J. Nutr., v.91,
p.331-339, 2004.
CHANDRA, R.K. Nutrition and the immune system from birth to old age. Eur. J. Clin. Nutr.,
v.3, p.73-76, 2002.
CHARGÉ, S.B.P. Interleukine-4 et fusion des cellules musculaires. M/S., v.19 (12), p.1185-
1187, 2003.
FERREIRA E SILVA, W.T. et al. Perinatal Malnutrition Programs Sustained Alterations in
Nitric Oxide Released by Activated Macrophages in Response to Fluoxetine in Adult Rats.
Neuroimmunomodulation., v.16, p.219-227, 2009.
FOSCHINI, R.M.S.A.; RAMALHO, F.S.; BICAS, H.E.A. Myogenic satellite cells. Arq. Bras.
Oftalmol., v.67 (4), p.681-687, 2004.
HELMING, L.; GORDON, S. The molecular basis of macrophage fusion. Immunobiol, v.212,
p.785-793, 2008.
HELMING, L.; GORDON, S. Molecular mediators of macrophage fusion. Trends in Cell
Biology, v.19, p.514-522, 2009.
HORSLEY, V. et al. IL-4 acts as a myoblast recruitment factor during mammalian muscle
growth. Cell., v.113, p.483-494, 2003.
LANDGRAF, M.A.V. et al. Intrauterine undernutrition in rats interferes with leukocyte
migration, decreasing adhesion molecule expression in leukocytes and endothelial cells. J.
Nutr., v.135, p.1480-1485, 2005.
LUCAS, A. Long-term programming effects of early nutrition -- implications for the preterm
infant. J Perinatol., v.25 (Sup. 2), p.S2-S6, 2005.
MELO, J.F. et al. Effect of neonatal malnutrition on cell recruitment and oxidant-antioxidant
activity of macrophages in endotoxemic adult rats. Rev. Nutr., v.21, p. 683-694, 2008.
MORENO, J.L. et al. IL-4 promotes the formation of multinucleated giant cells from
macrophage precursors by a STAT6-dependent, homotypic mechanism: contribution of E-
cadherin. J. Leukoc. Biol., v.82, p.1542–1553, 2007.
OZANNE, S.E.; HALES, C.N. Early programming of glucose-insulin metabolism. Trends
Endocrinol Metab., v.13 (9), p.368-373, 2002.
TE PAS, M.F. et al. Influences of myogenin genotypes on birth weight, growth rate, carcass
weight, backfat thickness, and lean weight of pigs. J. Anim. Sci., v.77, p.2352-2356, 1999.
WILSON, S.J.; ROSS, J.J.; HARRIS, A.J. A critical period for formation of secondary
myotubes defined by prenatal undernourishment in rats. Development, v.102, p.815-821,
1988.
LISTA DE ILUSTRAÇÕES
FIGURAS:
Figura 1. Programação de macrófagos em um estado competente para a fusão......
Figura 2. Mecanismo molecular de fusão do macrófago .........................................
Figura 3. Fusão das células musculares esqueléticas...............................................
Figura 4. Grupos experimentais................................................................................
Figura 5. Coleta do lavado broncoalveolar...............................................................
Figura 6. Obtenção e cultura dos macrófagos..........................................................
Figura 7. Retirada dos músculos das patas dos ratos para cultura in vitro...............
Figura 8. Obtenção e cultura de células musculares esqueléticas............................
Artigo 1
Figure 1. Body weight of pups from mothers fed a low-protein diet (undernutrition,
casein 8%) or a control diet (casein 17%) during growth. Analysis was performed
every 5 days during lactation (n=9 in each group) and at 42, 60 and 90 days (n=3 in
each group). The values are presented as means + S.E.M. *p<0.05 compared with
control group using two-way ANOVA and Bonferroni’s post hoc test
…………………………...................................................................................................
Figure 2. Numbers of macrophages/well (0.33 cm2) after 4 days in culture (A:42-day-
old offspring; B:60-day-old offspring; C: 90-day-old offspring). Data were obtained
from LDH activity of adherent cells. Data are expressed as mean + S.E.M. n=18 in
each group.*p<0.05 unpaired Student’s t-test…………………………………………...
30
30
33
41
42
43
44
45
64
65
LISTA DE ILUSTRAÇÕES
FIGURAS:
Artigo 1
Figure 3. IL-4 production in the supernatants of macrophages from 60 day-old
offspring, cultured for 4 days. Results were normalized to 108 cells. Data are represent
as mean + S.E.M. n=6 in each group. *p<0.05 Mann-Whitney test…………………….
Figure 4. Immunoblots of E (pan) cadherins in muscle cells from 60-day-old rats.
Muscle cells from control (C1, C2, C3) and undernourished offspring (UN1, UN2,
UN3) were cultured for 4 days. The lysates were separated by 7.5% SDS PAGE.
Actin was used as control for gel loading. The amount of cadherin relative to that
of actin is shown below. NS non-significant……………………………………………..
Figure 5. IFN-γ production in the supernatants of macrophages from 42 day-old
offspring (A), 60 day-old offspring (B) and 90 day-old offspring (C) cultured for 4
days. Results were normalized to 107 cells. Data are represent as mean + S.E.M. n=6
in each group. *p<0.05 Mann-Whitney test……………………………………………..
Artigo 2
Fig. 1 Body weight of pups from mothers fed a low-protein diet (undernutrition,
casein 8%) or a control diet (casein :17%) during growth. Analysis was performed
every five days during lactation (n=9 in each group), and at 42, 60 and 90 d (n=3 in
each group). The values are presented as means + S.E.M. p < 0.05 compared with
control group using two-way ANOVA and Bonferroni’s post hoc test………………..
66
67
68
73
LISTA DE ILUSTRAÇÕES
FIGURAS:
Artigo 2
Fig. 2 Morphological observation of muscle cells in culture (a), and numbers of
muscle cells/well (0.33 cm2) (b) after 10 days in culture (60-day-old offspring).
Mothers were submitted to a control diet (C1, C2, C3) or a low-protein diet (UN1,
UN2, UN3) during lactation. a Cells were cultured in a differentiation medium (DM)
and observed at 10 days. Phase contrast objective 10x. Bar: 100 µm. b Data were
obtained from LDH activity of adherent cells. Data are expressed as mean + S.E.M.
n=18 in each group. *p<0.05 unpaired Student’s t-test. C, control group; UN,
undernourished group……………………………………………………………………
Fig. 3 Morphological analysis of muscle cells in culture (90 day old offspring).
Mother´s offspring were submitted to a control diet (C1, C2, C3) or a low-protein diet
(UN1, UN2, UN3) during lactation. Cells were cultured in a differentiation medium
(DM) and analyzed on the 10th
day. Phase contrast objective 10x. Bar:100
µm………………………………………………………….............................................
Fig. 4 Immunoblots of Pax7 and M-N (pan) cadherins in muscle cells – from 60-day-
old rats. Muscle cells from control (C1, C2, C3) and undernourished offspring (UN1,
UN2, UN3) were cultured for 4 days. The lysates were separated by 7.5% SDS PAGE.
Actin was used as control for gel loading. The amount of each factor relative to that of
actin is shown below. NS non-significant……………………………………………….
74
75
76
LISTA DE TABELAS
Tabela 1. Composição das dietas experimentais à base de caseína (17% nutrido e 8%
desnutrido).........................................................................................................................
Artigo 1
Table 1. Composition of the diets (control 17% and low-protein 8%)…………………
Artigo 2
Table 1. Composition of the diets (control 17% and low-protein 8%)………………....
40
69
71
LISTA DE ABREVIATURAS E SIGLAS
AIN Instituto Americano de Nutrição
AM Macrófagos alveolares
BAL Lavado broncoalveolar
C Controle
CGM Células gigantes multinucleares
CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico
D Desnutrido
DM Meio de diferenciação
FAK Quinase de adesão focal
FAO Organização das Nações Unidas para Agricultura e Alimentação
FM Meio de fusão
IFN-ɣ Interferon-ɣ
IGF Fator de crescimento semelhante à insulina
IL Interleucina
IL-4Rα Receptor IL-4α
LAFINNT Laboratório de Fisiologia da Nutrição Naíde Teodósio
LBA Lavado broncoalveolar
LDH Lactato desidrogenase
LIKA Laboratório de Imunopatologia Keizo Asami
LPS Lipopolissacarídeo
MA Macrófagos alveolares
MGC Células gigantes multinucleares
MRFs Fatores regulatórios miogênicos
N Nutrido
NFATc2 Fator nuclear de ativação de células T
NS Não significante
PBS Tampão fosfato-salina
P-caderina Caderina placentária
LISTA DE ABREVIATURAS E SIGLAS
PCR Proteína C reativa
PGM Meio de crescimento primário
SDRA Síndrome do desconforto respiratório agudo
UFPE Universidade Federal de Pernambuco
UMR 6600 Unidade de Biomecânica-Bioengenharia
UN Desnutrido
UTC Universidade de Tecnologia de Compiègne
SUMÁRIO
RESUMO
ABSTRACT
RÉSUMÉ
1. APRESENTAÇÃO........................................................................................................ 25
2. REVISAO DA LITERATURA.................................................................................... 27
3. HIPÓTESES.................................................................................................................. 37
4. OBJETIVOS.................................................................................................................. 38
4.1. Geral......................................................................................................................... 38
4.2. Específicos................................................................................................................ 38
5. MATERIAIS E MÉTODOS......................................................................................... 39
5.1. Desenho do estudo.................................................................................................... 39
5.2. Animais e dietas....................................................................................................... 39
5.3. Operacionalização da pesquisa................................................................................ 41
5.3.1 Peso corporal.................................................................................................. 41
5.3.2 Lavado broncoalveolar (LBA)........................................................................ 42
5.3.3 Cultura de macrófagos alveolares................................................................... 42
5.3.4 Isolamento e cultura de células musculares.................................................... 44
5.3.5 Western Blot................................................................................................... 46
5.3.6 ELISA............................................................................................................. 47
5.3.7 Número total de células.................................................................................. 47
5.4. Análise estatística.................................................................................................... 48
6. RESULTADOS.............................................................................................................. 49
6.1. ARTIGO 1
Long-term effects of a neonatal low-protein diet in rats on the number of macrophages
in culture and the expression/production of fusion proteins ..............................................49
SUMÁRIO
6.2. ARTIGO 2
Effect of a neonatal low-protein diet on the morphology of myotubes in culture and
the expression of key proteins that regulate myogenesis in young and adult rats............... 70
7. CONCLUSÕES ............................................................................................................. 78
8. RECOMENDAÇÕES/PERSPECTIVAS.................................................................. 79
9. REFERÊNCIAS ........................................................................................................... 80
ANEXOS............................................................................................................................ 88
Anexo A – Aprovação do Comitê de Ética em Experimentação Animal da UFPE
Anexo B – Documentação do Artigo 1
Anexo C – Documentação do Artigo 2
25
1. APRESENTAÇÃO
Este trabalho de tese em co-tutela foi realizado na Universidade de Tecnologia de
Compiègne (UTC - França) e na Universidade Federal de Pernambuco (UFPE - Brasil) no
âmbito do projeto: “Papel profilático da atividade física sobre as consequências da desnutrição
precoce na função miocitária e nas propriedades neuro-mecânicas do músculo esquelético”
(CAPES/COFECUB nº584/07). Na França, as atividades foram realizadas na unidade de
Biomecânica-Bioengenharia (UMR 6600) da UTC. No Brasil, no Laboratório de Fisiologia da
Nutrição Naíde Teodósio (LAFINNT) do Departamento de Nutrição da UFPE, no Laboratório
de Imunopatologia Keizo Asami (LIKA-UFPE) e no Centro de Pesquisas Aggeu Magalhães
(Fiocruz PE).
O interesse por esse estudo surgiu quando foram avaliados os dados obtidos da
Dissertação de Mestrado “Atividade oxidante-antioxidante de macrófagos alveolares em ratos
endotoxêmicos submetidos à desnutrição neonatal” apresentada por mim à banca examinadora
do Curso de Pós-graduação em Medicina Tropical da Universidade Federal de Pernambuco
(Melo, 2007). Dentre os achados da pesquisa, foi observado que a desnutrição neonatal,
mesmo seguida de reposição nutricional, comprometeu a resposta inflamatória pulmonar de
ratos adultos, diminuindo o recrutamento de células inflamatórias para o pulmão e a atividade
oxidante-antioxidante dos macrófagos alveolares. Assim, surgiu a curiosidade de realizar um
estudo envolvendo não só células imunes, mas que pudesse aprofundar os mecanismos
envolvidos na programação nutricional também com células não imunes como as células
musculares esqueléticas. Parece haver um maior envolvimento da disfunção de células
musculares esqueléticas e fusão de macrófagos durante os estados sépticos. Além disso,
despertou-se o interesse em entender melhor esses mecanismos também em ratos mais jovens
e não somente em ratos adultos.
Os resultados desta tese se encontram apresentados na forma de (02) artigos:
O Artigo 1: avalia o efeito da desnutrição neonatal sobre o número de macrófagos em
cultura e a expressão/produção de proteínas que regulam a fusão macrofágica em
ratos jovens e adultos. Tal artigo foi intitulado: “Long-term effects of a neonatal low-
protein diet in rats on the number of macrophages in culture and the
26
expression/production of fusion proteins”. Enviado para publicação como artigo
original ao periódico British Journal of Nutrition.
O Artigo 2: Neste, foi avaliado o efeito da desnutrição neonatal sobre a morfologia de
miotubos em cultura e a expressão de proteínas-chaves que regulam a miogênese em
ratos jovens e adultos. Publicado como artigo original no European Journal of
Nutrition: MELO, J. F., Aloulou, Nijez, Duval, Jean-Luc, Vigneron, Pascale,
Bourgoin, Lee, Leandro, Carol Góis, Castro, Celia M. M. B., Nagel, Marie-Danielle.
Effect of a neonatal low-protein diet on the morphology of myotubes in culture and the
expression of key proteins that regulate myogenesis in young and adult rats. European
Journal of Nutrition 50: 243-250, 2011.
27
2. REVISÃO DA LITERATURA
2.1 Processo inflamatório/infeccioso: papel dos macrófagos
Os mecanismos de resistência do hospedeiro podem ser divididos em duas vias principais:
a resposta imune específica e a resposta imune inespecífica. As defesas inespecíficas incluem
a pele, as membranas mucosas, células fagocíticas, muco, epitélio ciliar, sistema
complemento, lisozima, interferon e outros fatores humorais (CHANDRA, 1997). Esses
processos inatos, do qual faz parte a reação inflamatória, estão naturalmente presentes, agem
como a primeira linha de proteção e retardam o estabelecimento de manifestações infecciosas
(CHANDRA, 1997).
A inflamação é definida clinicamente pela associação dos quatro sinais: tumor, rubor,
calor e dor. No que diz respeito aos tecidos, esses sinais são consequência de uma
vasodilatação, do aumento da permeabilidade vascular e de um afluxo de células fagocitárias
(DE CASTRO et al., 1997). No foco de injúria tecidual, fatores quimiotáticos atraem células
sanguíneas que, por diapedese, atravessam a barreira endotelial. Dentre estas células, os
neutrófilos estão em maior número, seguidos dos monócitos que se transformam em
macrófagos nos tecidos.
Os macrófagos são fagócitos que participam ativamente na defesa do hospedeiro frente às
infecções. Têm a capacidade de internalizar partículas inertes, células alteradas do indivíduo,
micro-organismos e parasitas. Dessa forma, a fagocitose constitui um dos principais
fenômenos que ocorrem no processo inflamatório.
Além de apresentarem uma alta capacidade fagocítica, os macrófagos são as células mais
frequentes dentre as que residem no pulmão (RUFINO e SILVA, 2006). São derivados da
medula óssea, pertencem ao sistema dos fagócitos mononucleares e constituem a segunda
maior população celular do sistema imune. Após o contato com o agente agressor, agem
liberando citocinas (MÁRTON e KISS, 2000) e outros potentes produtos microbicidas que
são responsáveis pela destruição dos micro-organismos fagocitados (AMERSFOORT et al.,
2003).
Em geral, os eventos que decorrem da sepse e do choque séptico são causados por
endotoxinas produzidas por bacilos Gram negativos. Essas substâncias se localizam na parte
28
externa da membrana bacteriana e são liberadas a partir da sua replicação e/ou morte (KLEIN
et al., 2011). A endotoxemia aguda, caracterizada por altos níveis de endotoxina no sangue,
causa reação inflamatória com injúria endotelial, hipotensão, falência múltipla dos órgãos e
morte (SUNIL et al., 2002).
Dentre os órgãos envolvidos no choque séptico, o pulmão é o primeiro a ser atingido
(D’ACAMPORA et al., 2001). Durante a endotoxemia, as complicações pulmonares,
incluindo edema pulmonar e falência respiratória, são a maior causa de morbi-mortalidade em
pacientes sépticos (WANG et al., 1994). O lipopolissacarídeo (LPS) ativa macrófagos e com
isto, induz a produção e liberação de inúmeras citocinas (MENG e LOWELL, 1997;
WEINSTEIN et al., 2000). Estas substâncias induzem a resposta de fase aguda da inflamação
e capacitam o sistema imune para uma atividade rápida frente aos processos infecciosos.
2.2 Fusão de macrófagos
Os macrófagos estão presentes em todos os tecidos do corpo e, no combate à infecção,
podem fusionar entre si em resposta à patógenos e materiais estranhos dando origem aos
osteoclastos (no osso) ou às células gigantes multinucleares (CGM) (em vários tecidos).
Também podem fusionar com células somáticas e tumorais (VIGNERY, 2005). Nas
alterações teciduais, os osteoclastos e as células gigantes exercem suas funções na osteoporose
e nas doenças inflamatórias crônicas, respectivamente. A formação destas últimas é conhecida
por aumentar a capacidade defensiva dos macrófagos (MACLAUCHLAN et al., 2009).
Os mecanismos moleculares que permitem a fusão dos macrófagos entre eles ainda são
pouco conhecidos (VIGNERY, 2005). Algumas moléculas têm sido implicadas nesse
processo o qual pode envolver a ação de glicoproteínas, como as caderinas, que medeiam a
fixação da membrana e fusão. Além disso, a fusão entre os macrófagos pode ser induzida por
mediadores solúveis como citocinas e fatores de crescimento (HELMING e GORDON, 2008).
As caderinas representam uma família de moléculas de adesão celular transmembranares,
dependentes do cálcio, que se ligam através de ligações homofílicas. São importantes não
apenas no estabelecimento e manutenção das uniões intercelulares, mas também na
determinação da especificidade adesiva das células (CAVALCANTE, 2008). Geralmente,
células com poucas moléculas de caderina apresentam menor adesividade. O cálcio é
29
essencial para a função adesiva das caderinas e na proteção contra a digestão pelas proteases
(WHEELOCK e JOHNSON, 2003; CAVALCANTE, 2008).
As caderinas são divididas em subclasses de acordo com sua distribuição tecidual. A
nomenclatura deriva-se do tecido no qual estas glicoproteínas foram inicialmente
identificadas. A E-caderina foi inicialmente identificada em células epiteliais. A caderina
placentária (P-caderina) foi encontrada inicialmente em placenta e no epitélio, embora possa
ser expressa em outros tecidos durante o desenvolvimento (CAVALCANTE, 2008). Os
anticorpos de amplo espectro anti-pan caderinas são capazes de detectar diversos tipos de
caderinas a partir de células em cultura.
Estudos mostram que a E-caderina contribui para a formação das CGM por intermédio da
citocina anti-inflamatória IL-4 (MORENO et al., 2007; HELMING e GORDON, 2009). Esta
citocina é secretada por várias células imunes (MARTINEZ et al., 2009), incluindo os
macrófagos (FERNANDEZ-BOYANAPALLI et al., 2009). Há mais de 10 anos já vem sendo
mostrado que a IL-4 induz a formação das CGM in vitro (McINNES e RENNICK, 1988;
McNALLY e ANDERSON, 1995). Moreno et al. (2007) demonstraram que a IL-4 contribui
para um aumento na expressão de E-caderina, e consequentemente, aumento da formação de
CGM in vitro.
Segundo Helming e Gordon (2009), para se tornarem multinucleares, os macrófagos
precisam adquirir um estado de competência para a fusão. Isso acontece através da ação de
estímulos endógenos e exógenos. Após se ligar ao receptor IL-4 de cadeia α, a IL-4 induz a
competência para a fusão através da ativação do fator de transcrição STAT6 como também
pelo contato macrófago-macrófago (Figuras 1 e 2) . Somente após isso é que se dá a adesão
entre as células mediada pela E-caderina (Figura 2). Além da IL-4, outras citocinas como IL-
13 e interferon-γ (IFN-γ) também podem induzir a formação das CGM (HELMING e
GORDON, 2008, 2009). Esta última tem um papel na ativação dos macrófagos, porém pouco
ainda se sabe sobre os mecanismos envolvidos nesse processo de fusão.
30
Figura 1. Programação de macrófagos em um estado
competente para a fusão. Fonte: Helming e Gordon, 2009
Figura 2. Mecanismo molecular de fusão do macrófago.
Fonte: Helming e Gordon, 2009
31
2.3 Infecção e comprometimento do músculo esquelético
Em condições normais, a massa muscular é mantida pelo balanço entre síntese e
degradação protéicas e a perda do músculo pode ocorrer em qualquer situação que perturbe
este equilíbrio. Durante a infecção e trauma grave a proteína muscular sofre proteólise, sendo
rapidamente mobilizada para atender às necessidades de defesa orgânica na síntese de
proteínas de fase aguda como a proteína C reativa (PCR), anticorpos e imunoglobulinas, entre
outros (BURET, 2008; FEFERBAUN et al., 2009).
A perda da massa muscular é comumente vista em pacientes sépticos e com febre.
Nestes, ocorre uma perda marcante de peso e de proteína corporal, além de uma perda de
músculo generalizada (HASSELGREN e FISCHER, 1998). Câncer e AIDS são doenças
associadas com perda significante do músculo, e o desenvolvimento da disfunção muscular
está associada a uma resposta deficitária ao tratamento (MUSCARITOLI et al., 2006;
SUPINSKI e CALLAHAN, 2007).
O prejuízo na capacidade funcional do músculo esquelético é uma das principais causas
de morbidade em pacientes com doenças agudas e crônicas tais como pneumonia, infecções
generalizadas que causam bacteremia, síndrome do desconforto respiratório agudo (SDRA),
uremia, trauma, entre outras (SUPINSKI e CALLAHAN, 2007). Nessas situações, a
deficiência nutricional pode contribuir para a perda do músculo e a inflamação sistêmica pode
ser o fator chave causador da disfunção do músculo esquelético. Os níveis circulantes de
várias citocinas estão aumentados em pacientes com sepse (BELPERIO et al., 2006;
CONRAADS, 2006).
Em animais experimentais, a administração de citocinas, como também a indução de
produção, têm sido implicadas em causar perda muscular (BUCK e CHOJKIER, 1996;
SUPINSKI e CALLAHAN, 2007). De acordo com Li et al. (2003), a incubação de linhagens
celulares musculares com citocinas causa uma perda de proteínas celulares. Estudos mostram
que a administração de Streptococcus pneumoniae (RUFF e SECRIST, 1984) ou endotoxina
de Escherichia coli (FRICK et al., 2008) é responsável pela perda significante de massa
muscular e também pela quebra mais rápida das proteínas musculares em ratos.
32
2.4 Células musculares esqueléticas e processo de fusão na formação da fibra
O sistema muscular esquelético constitui a maior parte da musculatura do corpo.
As células satélites musculares são uma população de células miogênicas, mononucleares e
indiferenciadas. Essas células estão presentes no músculo esquelético e estão associadas a
todos os tipos de fibras musculares. Quando cultivadas in vitro, podem ser caracterizadas
através da expressão do gene Pax7 (CHARGÉ e RUDNICKI, 2004). O Pax7 é um fator de
transcrição importante para o desenvolvimento do músculo e é expresso em células satélites
quiescentes e em proliferação (FOSCHINI et al., 2004). Após proliferarem em resposta a
estímulos fisiológicos, as células satélites dão origem à mioblastos que se diferenciam e
fusionam.
Os mioblastos, que são as células precursoras das fibras musculares esqueléticas, se
fusionam em duas fases. Na primeira fase (mioblasto x mioblasto), os mioblastos
diferenciados se fusionam para formação de um miotubo primário com um número limitado
de núcleos. Na segunda fase de fusão (mioblasto x miotubo), os mioblastos se fusionam com
os miotubos primários originando os miotubos secundários ou maduros (PAVLATH e
HORSLEY, 2003) (Figura 3).
Existem moléculas de adesão celular, como caderinas e integrinas, que são importantes
para a fusão das células musculares esqueléticas. As caderinas são proteínas
transmembranares dependentes do cálcio que conferem a adesão entre células vizinhas
(ANGST et al., 2001). As N e M caderinas são cruciais para a fusão dos mioblastos em
mamíferos. (MEGE et al., 1992; WRÓBEL et al., 2007).
As integrinas são proteínas de membrana envolvidas em diversos processos biológicos
como embriogênese, inflamação e agregação plaquetária (BUTERA WADSWORTH, 2003).
São proteínas heterodiméricas, originalmente conhecidas como receptores para moléculas da
matriz extracelular. Também medeiam a adesão célula-célula por se ligarem através de
ligações heterotípicas a membros da superfamília das imunoglobulinas (CAVALCANTE,
2008). De acordo com Schwander et al. (2003), as integrinas β1, que estão localizadas na
superfície dos mioblastos, exercem um papel importante na fusão dessas células.
Estudos vêm mostrando o papel de efetores que fazem parte da resposta imune sobre a
proliferação dos mioblastos (CHARGÉ, 2003). Em trabalhos realizados por Horsley et al.
33
(2003), confirmou-se a atuação da IL-4 sobre proliferação e fusão durante a regeneração
muscular. Trata-se da primeira demonstração da expressão de IL-4 por uma célula não imune.
O estudo de Horsley et al. (2003) implica a via de sinalização de NFATc2 (fator nuclear de
ativação de células T) como responsável pela ativação do gene interleucina 4 (IL-4) e
consequente fusão de células musculares (mioblasto x miotubo) através da interação desta
citocina, produzida pelo miotubo primário, com o seu receptor (IL-4Rα) presente sobre os
mioblastos. Segundo Chargé (2003), a mesma via de sinalização que ativa a fusão de células
musculares também atua na fusão de macrófagos, o que sugere a possibilidade de uma fusão
ilegítima entre macrófagos e miotubos.
A formação da fibra muscular também depende da presença de um fator de transcrição
chamado miogenina. Essa proteína é expressa em todos os mioblastos a partir do início da
diferenciação e sua expressão continua durante a fusão celular, marcando também o final da
proliferação dos mioblastos (TE PAS et al., 1999). De acordo com Isobe et al. (1998), a
expressão da miogenina é necessária em várias etapas da maturação, mesmo após a formação
do miotubo.
Figura 3. Fusão das células musculares esqueléticas. Fonte: Chargé, 2003
34
2.5 Desnutrição e sistema imune
A deficiência nutricional durante o período crítico de desenvolvimento dos sistemas de
homeostase, entre eles o sistema imune, é capaz de interferir na formação de processos
essenciais para a defesa do hospedeiro frente às infecções. Nessa etapa da vida, agressões
nutricionais poderão alterar o padrão de eventos celulares, com consequências deletérias tanto
na aquisição de padrões fisiológicos maduros do organismo (BORBA et al., 2000) quanto para
a ocorrência de eventos metabólicos (OZANNE e HALES, 2002; LUCAS, 2005).
Informações epidemiológicas mostram que a desnutrição protéica é um problema de saúde
pública que há tempos acomete grande parcela da população mundial, principalmente nos
países em desenvolvimento (GUBERNATIS, 2006; FAO, 2010). Segundo dados da
Organização das Nações Unidas para Agricultura e Alimentação (FAO), o número de pessoas
desnutridas no mundo chegou a 965 milhões em 2010 (FAO, 2010). No entanto, a evolução
do estado nutricional da população brasileira passou por um processo de transição nos últimos
anos. Ocorreu um declínio da prevalência de desnutrição infantil e elevação da prevalência de
sobrepeso/obesidade em adultos. (BATISTA FILHO e RISSIN, 2003). A desnutrição durante
o período crítico de desenvolvimento dos sistemas orgânicos vem sendo apontada como um
dos principais fatores não genéticos implicados na etiologia de doenças infecciosas e outras,
como por exemplo, diabetes tipo II e obesidade (BARKER, 2003). De acordo com Lucas
(2005), o termo programação vem sendo utilizado há alguns anos para explicar os eventos que
atuam num período crítico ou susceptível do desenvolvimento e as consequências na estrutura
ou função do organismo na vida adulta.
A desnutrição calórico-protéica está associada com a redução da imunidade mediada por
células, da função fagocítica, do sistema complemento, da produção de anticorpos e da
secreção e liberação de citocinas (CHANDRA, 2002). Nessa situação, os mecanismos de
defesa pulmonares são afetados com diminuição no número de macrófagos alveolares (MA)
(MELO et al., 2008; Ferreira e Silva et al., 2009). Além disso, várias funções dos macrófagos
encontram-se comprometidas (MELO et al., 2008; FERREIRA E SILVA et al., 2009). Estas
células, quando ativadas por endotoxinas, liberam produtos microbicidas que são responsáveis
pela destruição dos micro-organismos fagocitados (MEYER et al., 1994; AMERSFOORT et
al., 2003).
35
Tem aumentado o interesse dos cientistas pelos eventuais efeitos da privação de alimentos
sobre a função imune (ALLENDE et al., 1998). Dados epidemiológicos e clínicos sugerem
que deficiências nutricionais alteram a imunocompetência e aumentam o risco de infecções. A
desnutrição pode acarretar prejuízos na imunidade celular, mediada por células, e na
imunidade humoral, mediada pela produção de anticorpos, além de prejuízo nas funções dos
macrófagos e na atividade dos neutrófilos (CHANDRA, 1997).
Estudos realizados pelo nosso grupo de pesquisa (MELO et al., 2008; FERREIRA E
SILVA et al., 2009) demonstraram que a desnutrição precoce reduz o número de macrófagos
alveolares e compromete a atividade oxidante dessas células em ratos adultos. Várias funções
dos macrófagos encontram-se comprometidas na desnutrição (MELO et al., 2008;
FERREIRA E SILVA et al., 2009). Além disso, estudos mostram que a deficiência nutricional
causa uma redução na produção de anticorpos, na liberação de citocinas e na expressão de
moléculas de adesão celular (CHANDRA, 2002; LANDGRAF et al., 2005).
2.6 Desnutrição e suas repercussões no sistema muscular esquelético
Estudos recentes têm contribuído para o esclarecimento de alterações energéticas na
célula muscular decorrentes da desnutrição como, por exemplo, depleção enzimática,
disfunção mitocondrial, fosforilação oxidativa prejudicada e potencial de membrana celular
alterado (KRISTINA et al., 2005).
A desnutrição precoce é capaz de diminuir a capacidade de diferenciação de células
musculares e de reduzir o número de fibras presentes no músculo (WILSON et al., 1988;
BAYOL et al., 2004). Ratos cujas mães foram desnutridas com dieta à base de caseína 8%
apresentaram uma redução no número de fibras musculares e uma diminuição na formação de
miotubos secundários (WILSON et al., 1988). Ainda, Bayol et al. (2004) demonstraram que a
desnutrição no período gestacional reduziu a celularidade no músculo após 3 semanas de
reposição nutricional. Contudo, pouco tem sido pesquisado sobre o efeito da desnutrição
somente durante a lactação no desenvolvimento do músculo esquelético.
O processo miogênico é regulado por fatores de transcrição, como: Pax3/Pax7 e
fatores regulatórios miogênicos, como: MyoD, miogenina, Myf5 e Myf6 envolvidos
diretamente na formação dos músculos (COSSU et al., 1996). Segundo Halevy et al. (2003), a
36
restrição alimentar diminui a expressão dos marcadores da linhagem miogênica Pax7 e
miogenina. De acordo com Landgraf et al. (2005), ocorre uma redução da expressão de
moléculas de adesão celular em ratos adultos jovens que foram desnutridos no período
gestacional.
Apesar de existirem muitos estudos sobre o efeito da desnutrição sobre os sistemas
biológicos, ainda há muito a ser investigado a cerca do seu efeito tardio após reposição
nutricional sobre funções de células que atuam em conjunto no processo
inflamatório/infeccioso. Diante disso, a realização de trabalhos experimentais utilizando
modelos de desnutrição neonatal pode esclarecer os questionamentos a respeito do impacto
que este modelo pode causar na fusão de células macrofágicas e miocitárias no organismo
jovem e adulto. Isto pode auxiliar na compreensão do processo inflamatório/infeccioso nestes
organismos e, desta forma, contribuir na definição de estratégias adequadas para o controle
das doenças infecciosas.
37
3. HIPÓTESES
A desnutrição neonatal, seguida de reposição nutricional, causa na idade jovem e
adulta do animal:
Redução na expressão das moléculas de fusão Pan-caderina de macrófagos
alveolares e de células musculares esqueléticas;
Redução na expressão das moléculas de fusão β1-integrina e IL-4Rα em células
musculares esqueléticas;
Redução na produção de IL-4 por macrófagos alveolares e células musculares
esqueléticas;
Redução na produção de IFN-γ por macrófagos alveolares;
Redução na expressão dos fatores transcricionais Pax7 e miogenina (marcadores
da linhagem miogênica);
Redução do número de células totais através da diminuição na liberação de lactato
desidrogenase (LDH) por macrófagos alveolares e células musculares esqueléticas.
38
4. OBJETIVOS
4.1 Geral
Avaliar a expressão/produção de moléculas de fusão e fatores transcricionais em
macrófagos alveolares e células musculares esqueléticas de ratos jovens e adultos
submetidos à desnutrição neonatal.
4.2 Específicos
Comparar entre grupos de ratos nutridos e desnutridos no período neonatal
seguidos de reposição nutricional:
Expressão de Pan-caderina de macrófagos alveolares e de células musculares
esqueléticas em cultura;
Expressão de β1-integrina de mioblastos em cultura;
Expressão de IL-4Rα de mioblastos em cultura;
Níveis de IL-4 em sobrenadantes de cultura de macrófagos alveolares e miotubos;
Níveis de IFN-γ em sobrenadantes de cultura de macrófagos alveolares;
Expressão dos marcadores da linhagem miogênica Pax7 e miogenina;
Quantidade de células através dos níveis de lactato desidrogenase (LDH) em
sobrenadantes de cultura de macrófagos alveolares e de células musculares
esqueléticas.
39
5. MATERIAIS E MÉTODOS
5.1 Desenho do estudo
Trata-se de um estudo do tipo experimental. A escolha desse desenho metodológico
foi determinada pela grande comparabilidade entre os grupos em termos de variáveis de
confundimento e facilidade na formação do grupo controle. A comparabilidade é garantida
pela homogeneidade que os grupos apresentam quanto à idade, sexo, raça, tipo de dieta, etc.
5.2 Animais e dietas
Foram utilizados 18 ratos machos, albinos, da linhagem Wistar, provenientes da
colônia de criação do Departamento de Nutrição da Universidade Federal de Pernambuco
(UFPE) e 18 animais do Centre d’élevage Dépré (Saint-Doulchard, France). Os ratos foram
mantidos no biotério sob temperatura de 23 20C, ciclo claro-escuro de 12:12 h (com luzes
acesas às 6 h), recebendo ad libitum as respectivas rações e água até o início dos
experimentos.
Ratos com mais de 90 dias de idade foram acasalados pelo sistema poligâmico ou
harém, com a combinação de 1 macho/2 ou 3 fêmeas alojados em gaiolas de polipropileno
(49x34x16cm). Para obtenção dos animais experimentais, após o nascimento, o número de
filhotes por mãe foi padronizado em 6. Isto foi obtido reduzindo-se as proles ou
acrescentando-lhes animais da mesma idade, provenientes de outras ninhadas de forma que os
filhotes fossem distribuídos aleatoriamente entre as ninhadas do estudo. Após o nascimento
dos animais, estes foram divididos em 2 grupos de acordo com a dieta materna durante os
primeiros 21 dias pós-natais (período de aleitamento): Nutrido (N) (n=9) com dieta
normoprotéica à base de caseína a 17% (AIN 93G) (Tabela 1), e Desnutrido (D) (n=9) com
dieta experimental hipoprotéica à base de caseína a 8% (AIN 93G) (Tabela 1). Após o
desmame, os filhotes foram separados de suas mães, mantidos em gaiolas coletivas (contendo
3 animais cada), e receberam a dieta do biotério (LABINA – Purina do Brasil S/A ou
40
TEKLAD GLOBAL – Harlen Teklad) até completarem 42 dias (n=6), 60 dias (n=6) e 90 dias
de vida (n=6) (Figura 4).
Tabela 1. Composição das dietas experimentais à base de caseína (17% nutrido e
8% desnutrido)
Ingredientes Quantidade para 1 Kg de dieta
8% 17%
Caseína 79,3 g 179,3 g
Mistura vitamínica 10 g 10 g
Mistura mineral 35 g 35 g
Celulose 50 g 50 g
Bitartarato de colina 2,5 g 2,5 g
DL-Metionina 3,0 g 3,0 g
Óleo de soja 70 mL 70 mL
Amido de milho 750,2 g 650,2 g
* Composição das dietas segundo a AIN-93G – Reeves et al., 1993
41
Figura 4- Grupos experimentais
5.3 Operacionalização da pesquisa
5.3.1 Peso Corporal
Durante os primeiros 21 dias após o nascimento, foram registrados a cada cinco dias
os pesos corporais de cada animal, a fim de acompanhar o peso durante a manipulação
nutricional. A partir do 22 dia de vida até o final do experimento, o peso foi aferido 1 vez
por semana, objetivando acompanhar a evolução ponderal durante a fase de reposição
nutricional dos animais.
Nutrido (N) Desnutrido (D)
18 ratos machos
3
3
3
3
3
3
42 dias
60 dias
90 dias
17% caseína 8% caseína
LABINA ou
TEKLAD GLOBAL
ALEITAMENTO% caseína
42
5.3.2 Lavado broncoalveolar (LBA)
O lavado broncoalveolar foi obtido de acordo com a técnica utilizada por De Castro et
al. (1995). Os animais foram anestesiados com ketamina (45,2 mg/kg) e xilazina (6,8 mg/kg)
via intramuscular. Em seguida, o LBA foi coletado com a injeção de salina a 0,9% através de
uma cânula plástica inserida na traquéia (Figura 5). Várias alíquotas de 3 mL foram injetadas
e coletadas em tubos cônicos de polipropileno de 50 mL (Falcon, Sigma). Foi recuperado
aproximadamente 30 mL de LBA para cada animal.
Figura 5- Coleta do lavado broncoalveolar
5.3.3 Cultura de macrófagos alveolares
O LBA recolhido foi centrifugado a 450 g durante 15min. O precipitado que
corresponde às células foi ressuspendido em meio de cultura (RPMI 1640, Gibco-Invitrogen
Corporation) contendo soro fetal bovino (3%, Gibco-Invitrogen Corporation) e antibióticos
(penicilina 100U/mL e estreptomicina 100µg/mL). As células foram transferidas para placas
de cultura de 35 mm de diâmetro (6 poços, Falcon), onde foram dispensados 2 mL da
43
suspensão em uma proporção de 8x105
células/mL de RPMI 1640 em cada poço. Em placas
de cultura de 96 poços (0,33cm2/poço), foram depositadas 8x10
3 células/poço em um volume
final de 100µL de RPMI 1640 para determinação do número total de células. Após 1h na
incubadora a 37°C e 5% CO2, o sobrenadante com as células não aderentes foi desprezado e
foi adicionado 2 mL (placas 6 poços) e 100µL (placas 96 poços) de meio RPMI deixando-se
as placas por 4 dias em incubadora (Figura 6). O meio RPMI foi trocado a cada 2 dias
durante esse período de cultura, e os macrófagos foram estimulados in vitro com LPS
(sorotipo de Escherichia coli; 055:B5, Sigma) na dose de 10µg/mL em NaCl a 0,9% por 24
horas no 3º dia de cultura.
Queirós-Santos, 2000
Incubação à 37 C, 5% CO2
durante 4 dias
1.600.000 células/poço
Câmara de Neubauer
No 3 dia de cultura, as células foram
estimuladas in vitro com LPS (sorotipo
de Escherichia coli; 055:B5, Sigma)
na dose de 10µg/mL de NaCl a 0,9%
por 24 horas.8.000 células/poço
Figura 6- Obtenção e cultura dos macrófagos
44
5.3.4 Isolamento e cultura de células musculares
Todos os músculos foram retirados de ambas as patas dos animais (Figura 7) e
isolados em 15 mL de tampão fosfato-salina (PBS) contendo 0,15g de glicose e 1% de
solução antibiótica. Os ratos foram anestesiados com ketamina (45,2 mg/kg) e xilazina (6,8
mg/kg) via intramuscular.
Figura 7- Retirada dos músculos das patas dos ratos para cultura in vitro
Um dia antes do experimento, placas de cultura de 80cm2
(Nunclon®) foram cobertas
com 2 mL de Matrigel®
(BD Biosciences, Le Pont de Claix, France) diluído 1:500 em meio de
cultura DMEM (1g/L glicose) e incubadas à 37°C e 10% CO2. Os músculos foram cortados
em pequenos pedaços e, com o auxílio de tesouras ou bisturis, a gordura e os tendões
removidos. Os pedaços de músculo foram triturados e então colocados em 50 mL de meio de
crescimento primário (PGM) contendo DMEM (1g/L glicose), soro fetal bovino à 10%, soro
de cavalo à 10%, 4mMol/L de L-glutamina e 1% de solução antibiótica e centrifugados a 200
g durante 4min à 4°C. Os sobrenadantes foram coletados e o precipitado ressuspendido em
15mL de DMEM (1g/L glicose) suplementado com 0,03g de colagenase tipo II e 1% de
solução antibiótica, e incubados durante 90 min à 37°C sob agitação. Esta operação foi
repetida duas vezes utilizando-se 15 mL de DMEM (1g/L glicose) suplementado com 2 mL
de tripsina a 0,25% sem EDTA, 2mL de DNAse 1mg/mL de PBS (Sigma) e 1% de solução
45
antibiótica em adição de 0,04g de colagenase. Os precipitados foram então misturados com os
sobrenadantes coletados e filtrados (filtros 50µm, D Deutscher, Issy les Moulineaux, France).
As células foram contadas em Câmara de Malassez e transferidas para as placas de cultura na
proporção de 1,6x106 células/placa de 80cm
2 com volume final de 10 mL de PGM. Em placas
de cultura de 96 poços (0,33cm2/poço), foram depositadas 8x10
3 células/poço em um volume
final de 100µL de PGM para determinação do número total de células. Após 48h na
incubadora a 37°C e 10% CO2, o PGM foi substituído pelo meio de fusão (FM) contendo
DMEM (1g/L glicose), soro de cavalo à 7%, 4mMol/L de L-glutamina e 1% de solução
antibiótica. Esse meio foi trocado a cada 2 dias durante um período de cultura de 10 dias
(Figura 8). Todos os produtos utilizados para cultura de célula muscular, exceto o Matrigel e
a DNAse, foram da marca Gibco (Invitrogen, Cergy-Pontoise, France).
Incubação à 37 C, 10%
CO2 durante 10 dias
1.600.000
células/placa
Câmara de Malassez
8.000
células/poço
Figura 8- Obtenção e cultura de células musculares esqueléticas
46
5.3.5 Western Blot
Os precipitados dos macrófagos alveolares e das células musculares foram guardados à
-80°C e utilizados para avaliação da expressão de Pan-caderina (em macrófagos) e de Pan-
caderina, β1-integrina, IL-4Rα, Pax7 e miogenina (em células musculares) através da técnica
de Western Blot. Foram utilizados precipitados de macrófagos estimulados in vitro com LPS
por 24 horas e precipitados de células musculares cultivadas por 10 dias (0, 4, 7 e 10 dias).
Para os macrófagos, a expressão de Pan-caderina foi avaliada no dia 4 de cultura. Para as
células musculares, a expressão de Pax7 foi avaliada nos dias 0 e 4 de cultura; Pan-caderina
foi avaliada no dia 4 de cultura; β1-integrina nos dias 4 e 7; IL-4Rα no dia 7 e a miogenina no
dia 10 de cultura.
As células foram lisadas com tampão de lise (50 mM HEPES, 150 mM NaCl, 10%
glicerol, 1% Triton X 100, 1M NaF, 100 mM PMSF, 10mg/ml aprotinina, 10 mM
orthovanadato). Alíquotas dos sobrenadantes foram usadas para medir a concentração da
proteína total, como descrito por Bradford (1976). Os lisados protéicos das células foram
misturados com tampão Laemmli e fervidos durante 5 min. Quantidades iguais de proteínas
de cada amostra (40 μg para as células musculares e 160 μg para os macrófagos) foram
separadas no SDS PAGE (géis 7,5% ou 12%) e transferidas para membranas de nitrocelulose
(BioRad, Marnes-la-Coquette, France). Os filtros de nitrocelulose foram embebidos em
solução bloqueadora (PBS contendo 0,1% Tween 20 e 5% de leite desnatado) à temperatura
ambiente por 1h e em seguida incubados overnight à 4°C com os anticorpos anti-coelho anti-
Pax7 (Santa Cruz Biotechnology, Tebu-Bio, Velizy, France), anti-pan-caderina (Sigma, Saint-
Quentin Fallavier, France), ou anti-IL-4Rα (Santa Cruz Biotechnology, Tebu-Bio, Vélizy,
France), ou o anticorpo anti-β1 integrina de cabra (Santa Cruz Biotechnology, Tebu-Bio,
Vélizy, France). As membranas foram então incubadas com anticorpo anti-IgG de coelho
conjugado à HRP ou anticorpo anti-IgG de cabra ligado à peroxidase (Jackson
ImmunoResearch, Interchim, Montluçon, France) durante 3h à temperatura ambiente (para os
macrófagos) ou 1h (para as células musculares). As membranas foram incubadas durante 1h à
temperatura ambiente com o anticorpo monoclonal anti-miogenina de camundongo (BD
Biosciences, Le Pont de Claix, France) seguido por um anticorpo anti-IgG de camundongo
conjugado à HRP (Jackson ImmunoResearch, Interchim). A imunoreatividade foi detectada
47
através de quimioluminescência (ECL e hiperfilme ECL, GE Healthcare Europe, Saclay,
France). Os pesos moleculares das bandas imunoreativas foram estabelecidos através de
comparação com os padrões corados (BioRad, Marnes-la-Coquette, France; Amersham full-
range rainbow molecular weight markers, GE Healthcare, USA). Foram realizados Western
blots para as proteínas actina e α-tubulina com o intuito de checar o carregamento do gel. As
quantidades de proteínas com relação à actina ou α-tubulina foram determinadas e as
intensidades das bandas quantificadas usando-se o software de análise de imagem KODAK
1D (Scientific Image System, Rochester, NY, USA).
5.3.6 ELISA
As produções de IL-4 e IFN-γ foram avaliadas pelo método de ELISA utilizando-se o
kit ELISA DuoSet (R&D Systems). Amostras de 100μL foram coletadas a partir do
sobrenadante de cultura de macrófagos estimulados por 24 horas com LPS ou a partir do
sobrenadante de células musculares cultivadas por 10 dias. As concentrações dessas citocinas
foram normalizadas de acordo com a dosagem de LDH.
5.3.7 Número total de células
A quantidade total de células foi avaliada de forma indireta através da liberação da
enzima lactato desidrogenase (LDH) em sobrenadantes de células cultivadas em placas de 96
poços (0,33 cm2/poço). As células macrofágicas estimuladas com LPS por 24 horas e as
células musculares cultivadas por 10 dias foram lisadas em meio de cultura sem soro e
transferidas para outras placas de cultura de 96 poços. A liberação de LDH foi quantificada
com o kit de avaliação de citotoxicidade CytoTox 96 Non-Radioactive (Promega
Corporation), seguindo-se as instruções do fabricante. A absorbância (490nm) do vermelho de
formazan produzido pela LDH foi medida em um leitor de microplaca (Modelo 680, Bio-Rad)
e foi proporcional ao número total de células.
48
5.4 Análise Estatística
Para comparação de peso corporal, níveis de LDH e níveis das citocinas IL-4 e IFN-γ
entre os grupos, foi empregada a Análise de Variância (ANOVA), para os dados paramétricos.
Quando a ANOVA revelou a existência de diferença significativa, foi utilizado o Teste de
Tukey, a fim de identificar que grupos diferiram entre si. Para os dados não paramétricos, foi
utilizado o Teste de Kruskal-Wallis, seguido do Teste de Mann-Whitney. A significância
estatística foi considerada admitindo-se um nível crítico de 5% em todos os casos.
49
6. RESULTADOS
6.1 Artigo 1
Title of paper: Long-term effects of a neonatal low-protein diet in rats on the number of
macrophages in culture and the expression/production of fusion proteins
Names of authors:
Juliana Félix de Melo1,3,4*
Thacianna Barreto da Costa1,3
Tamara D. da Costa Lima2
Maria E.C. Chaves3
Muriel Vayssade 4
Marie-Danielle Nagel4
Célia M. M. B de Castro1,3
Affiliations of authors
1 Department of Tropical Medicine, Federal University of Pernambuco, Recife, Brazil
2 Aggeu Magalhães Research Center, Oswaldo Cruz Foundation, Campus UFPE, Recife,
Brazil
3 Keizo Asami Laboratory of Immunopathology, Federal University of Pernambuco, Recife,
Brazil
4 Université de Technologie de Compiègne, UMR CNRS 6600, Compiègne, France
*Address for correspondence:
Juliana Félix de Melo
Laboratório de Imunopatologia Keizo-Asami da Universidade Federal de Pernambuco (LIKA-
UFPE), Av. Prof. Moraes Rego, 1235, Cidade Universitária, 50670-901, Recife, Pernambuco,
Brazil.
Phone: +55 81 21268484
Fax: +55 81 21268485
E-mail: [email protected]
Running Title of paper: Macrophage fusion in undernourished rats
Keywords: Critical period of development; Programming; Macrophage fusion; Neonatal
malnutrition
50
Abstract
We investigate the effects of a neonatal low-protein diet on the number of macrophages in
culture and the expression/production of proteins that regulate macrophage fusion in young
and adult rats. Male Wistar rats (n=18) were suckled by mothers fed diets containing 17%
protein (controls), or 8% protein (undernourished, UN). All rats were fed a normal protein diet
after weaning. Bronchoalveolar lavage was collected from 42-, 60-, and 90-days-old rats.
Alveolar macrophages were cultured for 4 days in order to assess the number of cells and the
expression of cadherins, key proteins involved in macrophage fusion, by western blotting. IL-
4 and IFN-γ levels in culture supernatants were measured by ELISA. Offspring from mothers
fed a low-protein diet showed a lower body weight gain. The number of cells in cultured
macrophages from UN was evaluated on 42-, 60-, and 90-days-old rats; an increase on the
number of cells was observed on 42-, and 60-days-old rats, whereas a decrease was observed
on 90-days-old rats. Furthermore, IL-4 production was increased in the supernatants from UN
group of 60-days-old rats, but did not affect the expression of cadherins. IFN-γ production
increased in the supernatants from UN group of 42-, and 60-days-old rats, and decreased in
the later group. This study, thus, demonstrates that dietary restriction during lactation alters
the number of alveolar macrophages in culture, and the production of fusion proteins of
offspring aged 42, 60, or 90 days, but does not modify the expression of adhesion molecules,
which are important for the fusion of those cells.
51
Introduction
Malnutrition during the critical period of development of systems of homeostasis,
including the immune system, may impair the formation of processes essential for host
defense against infections. Changes at this stage in response to the environment can cause
permanent effects in organs and tissues, a phenomenon known as programming(1)
. At this
stage of life which includes prenatal and neonatal periods, nutritional assaults may alter the
pattern of cellular events, with deleterious consequences both for acquiring mature
physiological patterns of the organism(2)
and for the occurrence of metabolic events(1-3)
.
Experimental models in rats have shown that perinatal malnutrition may induce metabolic
diseases in adulthood(3,4)
.
Protein-calorie malnutrition is associated with a reduction in cell-mediated immunity,
phagocyte function, complement system, antibody production, cytokine release and
expression of cell adhesion molecules(5,6)
. In this situation, the pulmonary defense
mechanisms are affected with a resulting decrease in the number of alveolar macrophages
(AM)(7)
. In addition, several functions of macrophages are compromised(7)
. These cells, when
activated by endotoxins, release microbicide products that are responsible for the destruction
of phagocytosed microorganisms(8,9)
.
Macrophages are present in all body tissues and to fight infection, may fuse with each
other in response to pathogens and foreign materials, giving rise either to osteoclasts (bone) or
multinucleated giant cells (MGC) (in various tissues). The formation of MGC is known to
increase the defensive capacity of macrophages(10)
. The molecular mechanisms that allow the
fusion of macrophages with one another are still poorly understood(11)
. Some molecules have
been implicated in this process which may involve the action of glycoproteins such as
cadherins, which mediate membrane attachment and fusion. Studies show that IL-4
contributes to an increased expression of E-cadherin and, consequently, an increased
formation of MGC in vitro(12,13)
. After binding to the IL-4 receptor α chain, IL-4 induces
competence for fusion through the activation of the transcription factor STAT6 as well as by
macrophage-macrophage contact. It is only after this event that adhesion between cells
mediated by E-cadherin occurs. In addition to IL-4, other cytokines such as IL-13 and
interferon-γ (IFN-γ) can also induce the formation of the MGC(13,14)
. The latter has a role in
52
the activation of macrophages, but little is known about the mechanisms involved in this
fusion process.
Previous studies have reported the relationship between neonatal malnutrition and
impaired macrophage function in adulthood(7)
. However, much remains to be investigated
about its late effects, after nutritional recovery on macrophage fusion and on the behavior of
these cells in culture. In the present study, we evaluated the effects of neonatal malnutrition
on the number of alveolar macrophages in culture and the expression/production of proteins
that regulate the macrophage fusion in rats aged 42, 60 and 90 days.
Materials and methods
Ethical Considerations
This research was approved by the Ethics Committee on Animal Experimentation of
the Center for Biological Sciences of the Federal University of Pernambuco (protocol number
23076.005512/2008 – 40).
Animals and diet
Pregnant female Wistar rats were obtained from the Biotery of the Department of
Nutrition (Federal University of Pernambuco - UFPE, Brazil). The animals were housed under
conditions of controlled temperature (22 ± 1°C) in a 12h light-dark cycle. The standard animal
laboratory food and water were given ad libitum. At the time of delivery, the litter size and
pups’ birth weight were recorded. During the suckling period, the offspring were kept in
groups of six pups, randomly distributed into two nutritional groups according to their
mother’s diet: a well-nourished group (control, C) fed by mothers receiving a 17% protein
(casein) diet, and a low-protein group (undernourished, UN), fed by mothers receiving a 8%
protein (casein) diet (Table 1). After weaning (at the age of 22 days), only male offspring
were used (nine per group) and kept in the collective cage, receiving animal laboratory food
(Labina - Purina do Brasil) until the 90-day old ad libitum. Body weights were recorded every
five days (Filizola MF-3/1, São Paulo, SP) during lactation and at 42, 60 and 90 days. Body
weight gain was calculated as follows: percentage weight gain = [body weight (g) x
100/birthweight (g)] – 100 [11]. The animals were anesthetized with Ketamin (45.2
53
mg/kg)/Xylazin (6.8 mg/kg) by intramuscular injection at different ages (42, 60 and 90 days,
n=6 per age) and sacrificed after the procedures.
Bronchoalveolar lavage fluid (BAL)
The bronchoalveolar lavage was obtained according to techniques developed by De
Castro et al.(15)
. BAL was collected by injecting 0.9% NaCl through a plastic cannula inserted
into the trachea. Several aliquots of 3 mL were injected and collected in 50 mL conical
polypropylene tubes (Falcon, Sigma). Approximately 30 mL of BAL was recovered for each
animal.
Culture of alveolar macrophages (AM)
The collected BAL was centrifuged at 470 x g for 15 min. The precipitated part, which
correspond to the cells, was resuspended in a culture medium (RPMI 1640, Gibco-Invitrogen
Corporation, NY, USA) containing heat-inactivated fetal calf serum (3%, Gibco-Invitrogen
Corporation, NY, USA), penicillin (100 U/mL), streptomycin (100 µg/mL) and amphotericin
B (0.25 µg/mL) (Sigma). Cells were placed into 35cm2
and 0.33 cm2
tissue culture wells
(Falcon), where 2 mL (35cm2
well) and 100µL (0.33cm2
well ) of the suspension was
discarded in the proportion of 8 x 105
cells/mL of RPMI 1640 in each 35cm2
well and 8 x 103
cells/100µL of RPMI 1640 in each 0.33cm2
well. After being incubated for 1h at 37°C and
5% CO2, the supernatant containing nonadherent cells was discarded, and the remaining
monolayers (>95% AM) were incubated for an additional 1h in 2 mL (35cm2
well) and 100µL
(0.33cm2
well) of serum-free RPMI medium. After 3 days of culture, the cells were
stimulated in vitro with lipopolysaccharide (LPS) (sorotype from Escherichia coli 055:B5,
Sigma) at a dose of 10µg/mL of RPMI 1640, and 24h later the supernatants and cells
precipitates were collected for study. The number of cells was measured from 0.33cm2
wells
and the expression of proteins that regulate macrophage fusion from 35cm2
wells.
Number of cells
The number of cells was colorimetrically evaluated by measuring intracellular lactate
dehydrogenase (LDH) activity. Cells in culture for 4 days were lysed in serum-free RPMI
1640 and transferred to a 96-well plate. The LDH concentration was quantified with the
54
CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega Corporation, Madison, WI, USA)
in accordance with the manufacturer’s instructions. The absorbance (490nm) of the red
formazan produced by LDH was measured in a microplate reader (Model 680, Bio-Rad). The
absorbance was proportional to the total number of cells. LDH activity is given as the number
of cells, read off from a standard curve of absorbance plotted against an increasing number of
lysed cells.
Expression of Pan-cadherin
Proteins were extracted with lysis buffer (50 mM HEPES, 150 mM NaCl, 10%
glycerol, 1% Triton X 100, 1M NaF, 100 mM PMSF, 10mg/ml aprotinin, 10 mM
orthovanadate). Aliquots of supernatants were used for the measurement of total protein
content, as described by Bradford(16)
. Protein lysates from macrophages cultured for 4 days
were mixed with Laemmli buffer and boiled for 5 min. Equal amounts of proteins of each
sample (160 μg) were subjected to SDS PAGE (7.5% gels) and transferred to nitrocellulose
membranes (BioRad, Hercules, CA). The nitrocellulose filters were soaked in blocking
solution (PBS containing 0.1% Tween 20 and 5% non-fat dried milk) at room temperature for
1h and subsequently incubated overnight at 4°C with anti-pan-cadherin (Sigma, St. Louis,
USA). The membranes were then incubated with horseradish peroxidase-linked goat anti-
rabbit immunoglobulin G (Jackson ImmunoResearch, Interchim, Montluçon, France) for 3h at
room temperature. Immunoreactivity was detected by chemiluminescence (ECL and ECL
hyperfilm, GE Healthcare, Buckinghamshire, UK). The molecular weights of the
immunoreactive bands were established by comparison with stained standards (Amersham
full-range rainbow molecular weight markers, GE Healthcare, USA). Western blots for actin
were used to check gel loading. The ratios of proteins to actin were determined and band
intensities were quantified using KODAK 1D Image Analysis Software (Scientific Image
System, Rochester, NY, USA).
Concentrations of IL-4 and IFN-γ in the supernatants of macrophages in culture
The interleukin-4 (IL-4) and interferon-γ (IFN-γ) released were measured by ELISA in
the supernatants from cells cultured for 4 days (DuoSet ELISA Kit, R&D Systems,
55
Minneapolis, MN, USA). The IL-4 and IFN-γ concentrations were normalized to 108
and 107
cells, respectively, using the LDH dosage.
Statistical analysis
The data are presented as mean ± S.E.M. For body weight analysis two-way repeated
measures analysis of variance ANOVA, followed by Bonferroni’s post hoc test was used. For
LDH, IL-4 and IFN-γ levels in the supernatants of cultured cells and immunoblots
quantitation groups were compared using Student's t-test for parametric data and Mann-
Whitney test for nonparametric data. Results were considered statistically significant for
p < 0.05 (NS non-significant, *p<0.05). The GraphPad Prism 5 software (Graph Pad
Software, Inc., San Diego, CA, USA) was used.
Results
The birth weight of neonates did not differ when groups were compared (C = 6.5 ±
0.24 g and UN = 6 ± 0.27 g). During lactation, offspring from mothers fed a low-protein diet
showed a lower body weight than controls at 5, 10, 15 and 21 days. The pups from
undernourished mothers continued to exhibit a lower body weight than the control group
throughout the experiment: 42 d (C = 187.16 ± 9.16 g; UN = 161.33 ± 1.36 g), 60 d (C = 275
± 10.83 g; UN = 233.5 ± 5.25 g), and 90 d (C = 360 ± 12.05 g; UN = 300.33 ± 3.92 g) (Figure
1).
After 4 days of culture, LDH measurement showed a lower number of cells in the UN
when compared with cells from the C group at 42 d (C group 9.32 ± 0.99 x 103, UN group
2.22 ± 0.15 x 103) (Figure 2A) and 60 d (C group 3.48 ± 0.17 x 10
3, UN group 0.79 ± 0.18 x
103) (Figure 2B). At 90 d, however, there was an increase in the number of cells in the UN
group (C group 2.18 ± 0.12 x 103, UN group 3.51 ± 0.33 x 10
3) (Figure 2C). A lower number
of cells was observed with the age of the C offspring.
The concentration of IL-4 in the supernatants of alveolar macrophages cultured for 4
days, normalized using LDH activity, was increased in the UN group from 60-day-old
offspring (C group 54.58 ± 15.70 pg/mL x 108, UN group 267.44 ± 46.50 pg/mL x 10
8)
(Figure 3). There were no differences between UN and C offspring at 42 d and 90 d.
56
The cadherin (E) content of cells cultured for 4 days was assayed using a pan-cadherin
antibody. The amounts in 42-, 60- and 90-day-old pups appeared to be similar. There was no
difference in the expression of cadherin in cells from UN and C pups (Figure 4).
The production of IFN-γ in the supernatants of alveolar macrophages after 4 days of
culture, normalized using LDH activity, was increased in the UN group from 42-day-old
offspring (C group 21.11 ± 0.02 pg/mL x 107, UN group 88.88 ± 0.08 pg/mL x 10
7) (Figure
5A) and 60-day-old offspring (C group 56.46 ± 0.09 pg/mL x 107, UN group 251.22 ± 0.50
pg/mL x 107) (Figure 5B). Nevertheless, at 90 d, IFN-γ levels decreased in the UN group (C
group 90.48 ± 0.13 pg/mL x 107, UN group 56.27 ± 0.07 pg/mL x 10
7) (Figure 5C).
Discussion
Nutritional deficits are one of the most studied programming factors acting in the
critical period of development(17-19)
. Studies have shown that perinatal malnutrition acts in the
genesis of metabolic diseases such as obesity, diabetes, and cardiovascular diseases in
adulthood(19-22)
. In our study, casein (a highly phosphorylated protein) was chosen because it
is an important component of breast milk and has been widely used in previous studies as an
inducer of perinatal malnutrition.
In this study, we first examined the effects of a low-protein diet on the body weight of
the offspring during lactation. We then evaluated the number of alveolar macrophages in
culture and the expression/production of proteins known to regulate macrophage fusion, from
macrophage cultures of young and adult offspring. The results indicate that offspring from
mothers fed a low-protein diet showed a lower body weight during the lactation period from
the fifth day of life. These data are consistent with earlier studies using the same standard
experimental diet(19,23)
.
We also verified in this experiment the influence of a low-protein diet on the animals’
body weight at 42, 60 and 90 days. Our results show that even with the administration of a
balanced diet – Labina (bioterium diet containing 23% mixed protein) after weaning, the body
weight of the undernourished animals remained lower than that of the control animals. These
results are in agreement with other studies(23-27)
, in which even after nutritional recovery, the
body weight of undernourished animals remained low in adulthood. Passos et al.(28)
demonstrated that neonatal maternal protein restriction (8% casein-restricted diet) causes
57
changes in milk composition and volume of lactating rats, and it may be related to less
transfer of nutrients to the offspring and to a long-lasting low body weight.
Protein energy malnutrition leads to the depletion of progenitor hemopoietic
populations and changes in cellular development. Hemopoietic tissue requires a high nutrient
supply and the proliferation, differentiation and maturation of cells occur in a constant and
balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem
cell population(29)
. Macrophages are cells produced by the differentiation of monocytes in
tissues. These latter cells are produced by the bone marrow from haematopoietic stem cell
precursors called monoblasts. In the present study, we observed changes in the number of
macrophages after 4 days in culture when comparing UN and C offspring. There were fewer
macrophages in samples from 42 and 60-day-old UN pups. Nevertheless, in 90-day-old pups
it was observed that there was an increase of the macrophage number in the UN group,
suggesting that the experimental model used in this study is able to establish a compensatory
mechanism, significantly impairing the proliferative capacity of these cells in vitro. In
samples from C pups, a lower cell number was observed with the age of the offspring.
Previous studies(7,25)
have shown that neonatal malnutrition, even after nutritional recovery,
reduces the number of alveolar macrophages in adult rats. However, in those studies,
macrophages were counted from bronchoalveolar lavage fluid and were not maintained in
culture. In a previous study(24)
, using muscle cells from young and adult rats submitted to
neonatal malnutrition, a reduction in the number of cells was demonstrated in samples from
60-day-old UN pups after 10 days in culture, but no differences were observed in those from
90-day-old UN pups. Our results thus indicate that more studies are needed to evaluate the
behavior of these cells in vitro.
In order to test the hypothesis that neonatal malnutrition can impair the formation of
multinucleated giant cells (MGC), the capacity of alveolar macrophages from offspring of
different ages (42, 60 and 90 days) to produce fusion proteins in culture was assessed. Studies
show that cell adhesion molecules such as E-cadherins contribute to the formation of MGC
through IL-4(12,13)
. This cytokine is secreted by various immune cells(30)
, including
macrophages(31)
. It has been known for over 10 years that IL-4 induces the formation of MGC
in vitro(32,33)
, and it was recently demonstrated that IL-4 contributes to an increased expression
of E-cadherin and, consequently, to an increase in the formation of MGC in vitro(12,13)
. The
58
adhesion between cells mediated by E-cadherin occurs only after the fusion competence
induced by IL-4 through the activation of the transcription factor STAT6. The antibodies of
broad spectrum anti-pan cadherin are able to detect different types of cadherins from cells in
culture.
In 42- and 90-day-old animals, the neonatal malnutrition did not affect the production
of IL-4 or E-cadherin expression in macrophages, indicating a normal formation of MGC in
vitro. As for the 60-day-old animals that were malnourished during lactation, we observed an
increased production of IL-4, but that did not contribute to an increased expression of E-
cadherin. This may suggest that there is no commitment in the formation of MGC of these
animals mediated by E-cadherin. In a previous study(34)
, using cells from the spleen, bone
marrow, and peritoneal cavity, it was demonstrated that the levels of IL-4 did not differ in
undernourished mice inoculated with LPS when compared to control animals. Other studies
show that malnutrition can increase the production of IL-4 in CD4+ and CD8
+ cells
(35) and in
peripheral blood(36)
from undernourished children. Regarding E-cadherin, a previous study(24)
shows that neonatal malnutrition did not impair the expression of cadherins in skeletal muscle
cells of young and adult rats and that the amounts of these molecules appeared to be similar,
regardless of the age of the offspring, as observed in the present study.
Lastly, we analysed the production of IFN-γ, which can also induce the formation of
the MGC(13,14)
. It was observed that a neonatal low-protein diet increased IFN-γ levels in the
supernatants of alveolar macrophages from 42- and 60-day-old pups. Nevertheless, at 90 d, it
was a reduction in the production of IFN-γ in the UN group. Our results suggest that this
increased production of IFN-γ observed in younger animals may have been a compensatory
mechanism resulting from the low amount of cells at these ages. IFN-γ is a key cytokine in the
development of type 1 immune responses, which are required for the elimination of
pathogens(37)
. Also, IFN-γ induces differentiation and activation of monocytes/macrophages
and enhances their microbicidal effector functions(35,38)
. IL-4 is a Th2 cytokine which
suppresses cellular immunity(39)
. The elevated levels of IL-4 observed in this study in 60-day-
old offspring indicate a modulatory mechanism to suppress the high production of IFN-γ in
younger animals. As a result, the modulation in the synthesis of IFN-γ started to be observed
in older animals (90-day-old) with a low production of this cytokine. Thus, due to a probable
existing modulatory mechanism, we cannot suggest that the increase in the production of IFN-
59
γ is related to an increased formation of MGC in vitro. A number of studies show that
malnutrition can reduce the production of IFN-γ in CD4+ and CD8
+ cells from undernourished
children(35)
and spleen cells from mice(40)
. However, little is known about late effects of early
malnutrition on the production of these cytokines by alveolar macrophages.
Conclusion
Thus, based on the results of this study and review of the literature, casein restriction
during lactation followed by the restoration of a normal protein diet at weaning has no impact
on the expression of cadherins by alveolar macrophages of offspring aged 42, 60 or 90 days.
Nevertheless, the number and the production of fusion proteins of macrophages in culture are
altered.
Acknowledgements
The authors wish to thank CAPES-COFECUB (grant 584/07) and the National
Council for Science and Technology (CNPq), Brazil, for their financial support.
Conflict of interest
The authors declare that they have no conflict of interest.
60
References
1. Lucas A (2005) Long-term programming effects of early nutrition -- implications for
the preterm infant. J Perinatol 25, Suppl. 2, S2-S6.
2. Borba JMC, Araujo MS, Picanco-Diniz CW et al. (2000) Permanent and transitory
morphometric changes of NADPH-diaphorase-containing neurons in the rat visual
cortex after early malnutrition. Brain Res Bull 53, 193-201.
3. Ozanne SE & Hales CN (2002) Early programming of glucose-insulin metabolism.
Trends Endocrinol Metab 13, 368-373.
4. Kind KL, Simonetta G, Clifton PM et al. (2002) Effect of maternal feed restriction on
blood pressure in the adult guinea pig. Exp Physiol 87, 469-477.
5. Chandra RK (2002) Nutrition and the immune system from birth to old age. Eur J
Clin Nutr 3, 73-76.
6. Landgraf MAV, Martinez LL, Rastelli VMF et al. (2005) Intrauterine undernutrition in
rats interferes with leukocyte migration, decreasing adhesion molecule expression in
leukocytes and endothelial cells. J Nutr 135, 1480-1485.
7. Ferreira e Silva WT, Galvão BHA, Ferraz Pereira KN et al. (2009) Perinatal
Malnutrition Programs Sustained Alterations in Nitric Oxide Released by Activated
Macrophages in Response to Fluoxetine in Adult Rats. Neuroimmunomodulation 16,
219-227.
8. Meyer J, Hinder F, Stothert Jr J et al. (1994) Increased organ blood flow in chronic
endotoxemia is reversed by nitric oxide synthase inhibition. J Appl Physiol 76, 2785-
2793.
9. Amersfoort ESV, Berkel TJCV, Kuiper J (2003) Receptors, Mediators, and
Mechanisms Involved in Bacterial Sepsis and Septic Shock. Clin Microbiol Rev 16,
379-414.
10. MacLauchlan S, Skokos EA, Meznarich N et al. (2009) Macrophage fusion, giant cell
formation, and the foreign body response require matrix metalloproteinase 9. J Leukoc
Biol 85, 617-626.
11. Vignery A (2005) Macrophage fusion: the making of osteoclasts and giant cells. JEM
202, 337-340.
12. Moreno JL, Mikhailenko I, Tondravi MM et al. (2007) IL-4 promotes the formation
of multinucleated giant cells from macrophage precursors by a STAT6-dependent,
homotypic mechanism: contribution of E-cadherin. J Leukoc Biol 82, 1542–1553.
61
13. Helming L & Gordon S (2009) Molecular mediators of macrophage fusion. Trends
Cell Biol 19, 514-522.
14. Helming L & Gordon S (2008) The molecular basis of macrophage fusion.
Immunobiol 212, 785-793.
15. De Castro CMMB, Bureau MF, Nahori MA et al. (1995) Modulation by
dexamethasone of phospholipase A2 activities in endotoxemic guinea pigs. J Appl
Physiol 79, 1271-1277.
16. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72,
248-254.
17. Weinman AR, Jorge SM, Martins AR et al. (2007) Assessment of vitamin A
nutritional status in newborn preterm infants. Nutrition 23, 454-460.
18. Passos MC, Ramos CF, Moura EG (2000) Short and long term effects of malnutrition
in rats during lactation on the body weight of offsprings. Nutr Res 20, 1603-1612.
19. Pires-de-Melo IH, Wanderley dos Reis F, Luz LS et al. (2009) Short- and long-term
effects of a neonatal low-protein diet in rats on the morphology of the larynx.
Nutrition 25, 855-860.
20. Sawaya AL, Dallal G, Solymos G et al. (1995) Obesity and malnutrition in a
Shantytown
population in the city of Sao Paulo, Brazil. Obes Res 3, 107S-115S.
21. Barker DJ (1999) Early growth and cardiovascular disease. Arch Dis Child 80, 305-
307.
22. Barker DJ (2007) The origins of the developmental origins theory. J Intern Med
261, 412-417.
23. Araújo FRG, De Castro CMMB, Rocha JA et al. (2011) Perialveolar bacterial
microbiota and bacteraemia after dental alveolitis in adult rats that had been subjected to
neonatal malnutrition. Br J Nutr (Epublication ahead of print version).
24. Melo JF, Aloulou N, Duval JL et al. (2011) Effect of a neonatal low-protein diet on the
morphology of myotubes in culture and the expression of key proteins that regulate
myogenesis in young and adult rats. Eur J Nutr 50, 243-250.
25. Melo JF, Macêdo EMC, Paes-Silva RP et al. (2008) Effect of neonatal malnutrition on
cell recruitment and oxidant-antioxidant activity of macrophages in endotoxemic adult
rats. Rev Nutr 21, 683-694.
62
26. Barros KMFT, Manhães-De-Castro R, Lopes-De-Souza S et al. (2006) A regional
model (Northeastern Brazil) of induced mal-nutrition delays ontogeny of reflexes and
locomotor activity in rats. Nutr Neurosci 9, 99-104.
27. Barreto-Medeiros JM, Feitoza EG, Magalhães K et al. (2004) Malnutrition during brain
growth spurt alters the effect of fluoxetine on aggressive behavior in adult rats. Nutr
Neurosci 7, 49-52.
28. Passos MC, Da Fonte Ramos C, Dutra SC et al. (2002) Long-term effects of
malnutrition during lactation on the thyroid function of offspring. Horm Metab Res 34,
40-43.
29. Borelli P, Barros FEV, Nakajima K et al. (2009) Protein-energy malnutrition halts
hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell
production rates. Braz J Med Biol Res 42, 523-530.
30. Martinez FO, Helming L, Gordon S (2009) Alternative activation of macrophages: an
immunologic functional perspective. Annu Rev Immunol 27, 451-483.
31. Fernandez-Boyanapalli RF, Frasch SC, McPhillips K et al. (2009) Impaired apoptotic
cell clearance in CGD due to altered macrophage programming is reversed by
phosphatidylserine-dependent production of IL-4. Blood 113, 2047-2055.
32. McInnes A & Rennick DM (1988) Interleukin 4 induces cultured
monocytes/macrophages to form giant multinucleated cells. J Exp Med 167, 598-611.
33. McNally AK & Anderson JM (1995) Interleukin-4 induces foreign body giant cells
from human monocytes/macrophages. Differential lymphokine regulation of
macrophage fusion leads to morphological variants of multinucleated giant cells. Am J
Pathol 147, 1487-1499.
34. Fock RA, Vinolo MAR, Crisma AR et al. (2008) Protein-energy malnutrition modifies
the production of interleukin-10 in response to lipopolysaccharide (LPS) in a murine
model. J Nutr Sci Vitaminol 54, 371-377.
35. Rodríguez L, González C, Flores L et al. (2005) Assessment by flow cytometry of
cytokine production in malnourished children. Clin Diagn Lab Immunol 12, 502-507.
36. Villarroel MV, Valbuena AA, Pereira N et al. (2008) Citocinas TH2 (IL4 e IL10) en el
niño desnutrido. Arch Venez Puer Ped 71, 42-47.
37. Young HA & Hardy KJ (1995) Role of interferon-γ in immune cell regulation. J
Leukoc Biol 58, 373-381.
38. Chan J, Tanaka K, Mannion C et al. (1997) Effects of protein calorie malnutrition on
mice infected with BCG. J Nutr Immunol 5, 11-19.
63
39. Arababadi MK, Pourfathollah AA, Daneshmandi S et al. (2009) Evaluation of relation
between IL-4 and IFN-γ polymorphisms and type 2 diabetes. Iran J Basic Med Sci 12,
100-104.
40. Serafim TD, Malafaia G, Silva ME et al. (2010) Immune response to Leishmania
(Leishmania) chagasi infection is reduced in malnourished BALB/c mice. Mem Inst
Oswaldo Cruz 105, 811-817.
64
Figures and Legends
Figure 1 Body weight of pups from mothers fed a low-protein diet (undernutrition, casein
8%) or a control diet (casein 17%) during growth. Analysis was performed every five days
during lactation (n=9 in each group), and at 42, 60 and 90 d (n=3 in each group). The values
are presented as means + S.E.M. *p<0.05 compared with control group using two-way
ANOVA and Bonferroni’s post hoc test.
65
Figure 2 Numbers of macrophages/well (0.33 cm2) after 4 days in culture (A:42-day-old
offspring; B: 60-day-old offspring; C: 90-day-old offspring). Data were obtained from LDH
activity of adherent cells. Data are expressed as mean + S.E.M. n=18 in each group. *p<0.05
unpaired Student’s t-test.
66
Figure 3 IL-4 production in the supernatants of macrophages from 60 day-old offspring, cultured
for 4 days. Results were normalized to 108 cells. Data are represent as mean + S.E.M. n=6 in each
group. *p<0.05 Mann-Whitney test.
67
Figure 4 Immunoblots of E (pan) cadherins in muscle cells from 60-day-old rats. Muscle cells
from control (C1, C2, C3) and undernourished offspring (UN1, UN2, UN3) were cultured for 4
days. The lysates were separated by 7.5% SDS PAGE. Actin was used as control for gel loading.
The amount of cadherin relative to that of actin is shown below. NS non-significant.
68
Figure 5 IFN-γ production in the supernatants of macrophages from 42 day-old offspring (A), 60
day-old offspring (B) and 90 day-old offspring (C) cultured for 4 days. Results were normalized to
107 cells. Data are represent as mean + S.E.M. n=6 in each group. *p<0.05 Mann-Whitney test.
69
Table 1. Composition of the diets (control 17% and low-protein 8%)
Ingredients Amount for 1 Kg of diet
Low-
protein
Control
Casein 79,3 g 179,3 g
Vitamin Mix* 10 g 10 g
Mineral Mixture # 35 g 35 g
Cellulose 50 g 50 g
Choline bitartrate 2,5 g 2,5 g
DL-Methionine 3,0 g 3,0 g
Soya oil 70 ml 70 ml
Corn Starch 750,2 g 650,2 g # Mineral mixture contained the following (mg/kg of diet): CaHPO4, 17200; KCI, 4000; NaCl,
4000; MgO, 420; MgSO4, 2000; Fe2O2, 120; FeSO4·7H2O, 200; trace elements, 400
(MnSO4·H2O, 98; CuSO4·5H2O, 20; ZnSO4·7H2O, 80; CoSO4·7H2O, 0.16; KI, 0.32;
sufficient starch to bring to 40 g [per kg of diet]).
* Vitamin mixture contained the following (mg/kg of diet): retinol, 12; cholecalciferol, 0.125;
thiamine, 40; riboflavin, 30; pantothenic acid, 140; pyridoxine, 20; inositol, 300;
cyanocobalamin, 0.1; menadione, 80; nicotinic acid, 200; choline, 2720; folic acid, 10; p-
aminobenzoic acid, 100; biotin, 0.6.
6. RESULTADOS
6.2 Artigo 2 Eur J Nutr
DOI 10.1007/s00394-010-0132-9
OR IGIN A L C O N TR IB U TIO N
Effect of a neonatal low-protein diet on the morphology
of myotubes in culture and the expression of key proteins that regulate myogenesis in young and adult rats
Juliana Felix de Melo • Nijez Aloulou • Jean-Luc Duval • Pascale Vigneron •
Lee Bourgoin • Carol Go is Leandro • Celia M. M. B. de Castro • Marie-Danielle Nagel
Received: 6 April 2010 / Accepted: 30 August 2010
Springer-Verlag 2010
Abstract
Aim To investigate the effects of a neonatal low-protein
diet on the morphology of myotubes in culture and the
expression of key proteins that regulate myogenesis in
young and adult rats.
Methods Male Wistar rats (n = 18) were suckled by
mothers fed diets containing 17% protein (controls, C) or
8% protein (undernourished, UN). All rats were fed a
normal protein diet after weaning. Muscles were removed
from the legs of 42-, 60- and 90-day-old rats. Muscle cells
were cultured to assess cell number, morphology and the
expression of major proteins involved in myogenesis
(Pax7, cadherins, b1 integrin, IL-4Ra and myogenin) by
western blotting. IL-4 levels in culture supernatants were
measured by ELISA.
Results Offspring from mothers fed a low-protein diet
showed a lower body weight gain. Cell number and myo-
tube expansion were reduced in cultured muscle cells from
J. F. de Melo N. Aloulou J.-L. Duval P. Vigneron
L. Bourgoin M.-D. Nagel
Universite de Technologie de Compiegne,
UMR CNRS 6600, Compiegne, France
C. G. Leandro
Department of Physical Education and Sports Science, Centro
Academico de Vitoria, Federal University of Pernambuco,
Pernambuco, Brazil
J. F. de Melo C. M. M. B. de Castro
Department of Tropical Medicine,
Federal University of Pernambuco, Pernambuco, Brazil
J. F. de Melo (&)
Laborato rio de Imunopatologia Keizo-Asami (LIKA),
Setor de Microbiologia, UFPE, Cidade Universitaria, 50670-420 Recife, PE, Brazil
e-mail: [email protected]
UN, but the expression of myogenic marker proteins was
unaltered.
Conclusions Dietary restriction during lactation had no
impact on the synthesis of myogenic marker proteins, and
myocyte differentiation occurred normally in the muscles
of offspring aged 42, 60 or 90 days. Nevertheless, the
number and morphology of the myotubes are altered.
Keywords Critical period of development
Programming Satellite cells Neonatal undernutrition
Skeletal muscle Rats
Introduction
Maternal and neonatal undernutrition not only causes
prolonged growth retardation but also modifies the pro-
gramming of biochemical mechanisms related to endo-
crine-metabolic control [1]. Fetal programming is the
phenomenon whereby changes in the critical period of
development in response to the environment may have
permanent effects on the organs and tissues [2]. Perinatal
undernutrition-induced metabolic diseases in adult life
have been demonstrated in experimental models in rats [3,
4]. In humans, epidemiological studies have also revealed
an inverse relationship between birthweight and type 2
diabetes mellitus, hypertension, hyperlipidemia, obesity
and insulin resistance in adult life [5–7]. It has been
speculated that early influences on the growth and devel-
opment of skeletal muscle fibers may underlie this rela-
tionship [8].
Perinatal undernutrition on muscle-fiber development
and composition has been described in a variety of mam-
malian species [9]. Wilson et al. [10] showed a reduction in
secondary fibers in both soleus and lumbrical muscles and
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Eur J Nutr
a deficit in the growth rate of the newborn rat, when the
maternal diet was restricted to 30% of the control animals.
Maternal low-protein exposure (9% casein) during mid- to
late pregnancy reduces the numbers of fibers formed within
the soleus and gastrocnemius muscles of young rats [9]. On
the other hand, Bayol et al. [11] found no effect of maternal
undernutrition on numbers of fibers in the semitendinosus
muscle at 21 days postpartum but an increased muscle-cell
proliferation and a trend toward increased satellite-cell
activity.
Satellite cells are responsible for the growth, repair and
maintenance of the skeletal muscle [8]. In response to
environmental activating signals derived from injury,
satellite cells undergo asymmetric cell divisions for the
purpose of self-renewal, giving rise to committed myo-
genic cells, myoblasts, thereby sustaining postnatal muscle
repair and growth [8]. Adhesion molecules, growth factors
and cytokines released by the neighboring cells can also
activate satellite cells [12]. The myogenic process is reg-
ulated by transcription factors, such as Pax3/Pax7, and
members of the family of the myogenic regulatory factors
(MRFs), including MRF4 (Myf6), Myf5, MyoD and
myogenin [12].
Myogenin and MyoD may have important functions in
later development, since they bind directly to the enhancers
and activate transcription of the creatine kinase and myosin
light chain [8]. Several gene products are crucial for the
fusion of mammalian myoblasts, including cell adhesion
molecules (N and M cadherins) and the b1 integrins [13]. It
was recently demonstrated that newly formed myotubes
also secrete IL-4, which interacts with the IL-4 a receptors
(IL-4Ra) present on surrounding myoblasts to promote
their fusion with existing myotubes, thereby increasing
myotube size [14].
Previous studies have reported the relationship between
maternal undernutrition and the expression of genes that
control muscle growth in offspring at weaning [11]. Little
is known about the long-term effects on the satellite-cell
activity in adult rats, and the mechanisms by which
maternal nutrition may influence muscle growth. In the
present study, we evaluated the effects of neonatal under-
nutrition on the morphology of myotubes in culture and the
expression of proteins that regulate myogenesis in rats aged
42, 60 and 90 days.
Materials and methods
Animals and diet
Pregnant female Wistar rats were purchased from Centre
d’elevage Depre (Saint-Doulchard, France). Animals were
handled in accordance with the guidelines and regulations
of the Ethics and Safety Committee of the Compiegne
University of Technology and housed under conditions of
controlled temperature (22 ± 1 C), 12-h light–dark cycle.
The standard animal laboratory food and water were given
ad libitum. At the time of delivery, the litter size and pups’
birth weight were recorded. During the suckling period, the
offspring were kept in groups of six pups, randomly dis-
tributed into two nutritional groups according to their
mothers’ diet: a well-nourished group (control, C) fed by
mothers receiving a 17% protein (casein) diet and a low-
protein group (undernourished, UN), fed by mothers
receiving a 8% protein (casein) diet (Table 1). After
weaning (at the age of 22 days), only male offspring were
used (nine per group) and kept in the collective cage,
receiving standard animal laboratory food (Teklad Global
18% protein rodent diet; Harlen Teklad) until the 60-day-
old ad libitum. Subsequently, offspring were fed by a 14%
protein diet (Teklad Global 14% protein rodent diet; Harlen
Teklad) until the 90th day of life. Body weights were
recorded every 5 days (Mettler Toledo XS4001S, 0.1 g
readability, Inc, Columbus, OH) during lactation and at
42, 60 and 90 days. Body weight gain was calculated
as follows: percentage weight gain = [body weight (g) x
100/birthweight (g)] - 100 [11].
Offspring were anesthetized with Ketamin (45.2 mg/kg)/
Xylazin (6.8 mg/kg) by intramuscular injection at differ-
ent ages (42, 60 and 90 days, n = 6 per age). The muscles
from both legs were removed and immersed in ice-cold 15 mL
phosphate-buffered saline (PBS) containing 0.15 g glucose
Table 1 Composition of the diets (control 17% and low protein 8%)
Ingredients Amount for 1 kg of diet
Low protein Control Casein 79.3 g 179.3 g
Vitamin mixa
10 g 10 g
Mineral mixtureb
35 g 35 g
Cellulose 50 g 50 g
Choline bitartrate 2.5 g 2,5 g
DL-methionine 3.0 g 3,0 g
Soya oil 70 mL 70 mL
Corn starch 750.2 G 650.2 g
a Vitamin mixture contained the following (mg/kg of diet):
retinol,
12; cholecalciferol, 0.125; thiamine, 40; riboflavin, 30; pantothenic
acid, 140; pyridoxine, 20; inositol, 300; cyanocobalamin, 0.1; men-
adione, 80; nicotinic acid, 200; choline, 2,720; folic acid,
10; p-aminobenzoic acid, 100; biotin, 0.6 b Minerals mixture contained the following (mg/kg of diet):
CaHPO4,
17,200; KCI, 4,000; NaCl, 4,000; MgO, 420; MgSO4, 2,000; Fe2O2, 120; FeSO4 7H2O, 200; trace elements, 400 (MnSO4 H2O,
98; CuSO4 5H2O, 20; ZnSO4 7H2O, 80; CoSO4 7H2O, 0.16; KI, 0.32; sufficient starch to bring to 40 g [per kg of diet])
71
Eur J Nutr
and 1% antibiotic solution (Gibco, InVitrogen Cergy Pon-
toise, France). The animals were killed after the procedures.
Primary culture of rat skeletal muscle cells
All products for cell culture except Matrigel and DNAse
were from Gibco (InVitrogen, Cergy-Pontoise, France).
Primary culture of rat skeletal muscle cells was performed
as previously described [15]. Briefly, the soleus, gastroc-
nemius and quadriceps muscles were dissected, cut into
small pieces and digested with 2% type II collagenase,
0.25% trypsin and 0.1% DNAse (Sigma) for isolation of
myoblasts. Cells were placed into 80-cm2
Petri
dishes (Nunclon
) coated with 2 mL Matrigel
(BD
Biosciences, Le Pont de Claix, France) diluted 1:500 in
DMEM. Cells (1.6 x 106
cells/80-cm2
dishes) were
cultured in DMEM containing 10% fetal bovine serum,
10% horse serum,
4mMol/L L-glutamine and 1% antibiotic solution. After
48 h of culture, a differentiation medium (DM) with 7%
horse serum, 4mMol/L L-glutamine and 1% antibiotic
solution was used and changed every 2 days for a total of
10 days.
Cell number and morphology of the myotubes
in culture
Cell number was colorimetrically evaluated by measuring
intracellular lactate dehydrogenase (LDH) activity. Cells in
culture for 10 days were lysed in serum-free DMEM and
transferred to a 96-well plate. The LDH concentration was
quantified with the CytoTox 96 Non-Radioactive Cyto-
toxicity Assay (Promega Corporation, Madison, WI, USA)
in accordance with the manufacturer’s instructions. The
absorbance (490 nm) of the red formazan produced by
LDH was measured in a microplate reader (Model 680,
Bio-Rad). The absorbance was proportional to the total
number of cells. LDH activity is given as the number of
cells, read off from a standard curve of absorbance plotted
against an increasing number of lysed cells.
The morphology of myotubes in culture for 10 days was
observed using a contrast microscope (Olympus CKX41,
Japan) coupled to a digital camera (Canon A620). Quali-
tative analysis was performed from 5 fields by dish at 100x
magnification.
Expression of myogenic marker proteins
Proteins were extracted with lysis buffer (50 mM HEPES,
150 mM NaCl, 10% glycerol, 1% Tritonx 100, 1 M NaF,
100 mM PMSF, 10 mg/mL aprotinin, 10 mM orthovana-
date). Aliquots of supernatants were used for the mea-
surement of total protein content, as described by Bradford
[16]. Protein lysates from muscle cells cultured for 0, 4, 7
and 10 days were mixed with Laemmli buffer and boiled
for 5 min. Equal amounts of proteins of each sample
(40 µg) were subjected to SDS–PAGE (7.5% or 12% gels)
and transferred to nitrocellulose membranes (BioRad,
Marnes-la-Coquette, France). The nitrocellulose filters
were soaked in blocking solution (PBS containing 0.1%
Tween 20 and 5% non-fat dried milk) at room temperature
for 1 h and subsequently incubated overnight at 4 C with
rabbit anti-Pax7 (Santa Cruz Biotechnology, Tebu-Bio,
Velizy, France), anti-pan-cadherin (Sigma, Saint-Quentin
Fallavier, France), anti-IL-4Ra (Santa Cruz Biotechnology,
Tebu-Bio, Velizy, France) antibodies or a goat anti-b1
integrin antibody (Santa Cruz Biotechnology, Tebu-Bio,
Velizy, France). The membranes were then incubated with
horseradish peroxidase-linked goat anti-rabbit immuno-
globulin G or a peroxidase-linked donkey anti-goat
immunoglobulin G (Jackson ImmunoResearch, Interchim,
Montlucon, France) for 1 h at room temperature. Mem-
branes were incubated for 1 h at room temperature with
mouse monoclonal anti-myogenin antibody (BD Biosci-
ences, Le Pont de Claix, France), followed by a goat anti-
mouse IgG coupled to horseradish peroxidase (Jackson
ImmunoResearch, Interchim, France). Immunoreactivity
was detected by chemiluminescence (ECL and ECL
hyperfilm, GE Healthcare Europe, Saclay, France). The
molecular weights of the immunoreactive bands were
established by comparison with stained standards (BioRad,
Marnes-la-Coquette, France). Western blots for actin and
a-tubulin were used to check gel loading. The ratios of pro-
teins to actin or a-tubulin were determined, and band inten-
sities were quantified using KODAK 1D Image Analysis
Software (Scientific Image System, Rochester, NY, USA).
Concentration of IL-4 in the supernatants of muscle
cells in culture
Interleukin-4 (IL-4) released was measured by ELISA in
the supernatants from cells cultured for 10 days (DuoSet
ELISA Kit, R&D Systems, Minneapolis, MN, USA). The
IL-4 concentration was normalized to 105
cells using the
LDH dosage.
Statistical analysis
The data are presented as mean ± SEM. For body weight
analysis, two-way repeated measures analysis of variance
ANOVA, followed by Bonferroni’s post hoc test, was used.
For LDH and IL-4 levels in the supernatants of cultured
cells and immunoblots quantitation groups were compared
using Student’s t-test. Results were considered statistically
significant for p < 0.05 (NS non-significant, *p < 0.05).
The GraphPad Prism 5 software (Graph Pad Software, Inc.,
San Diego, CA, USA) was used.
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Eur J Nutr
Results
The birth weight of neonates did not differ when groups
were compared (C = 5.9 ± 0.4 and UN = 5.8 ± 0.3).
During lactation, offspring from mothers fed a low-protein
diet showed a lower body weight than controls at 10, 15
and 21 days. The pups from undernourished mothers con-
tinued to exhibit a lower body weight than the control
group throughout the experiment: 42 days (C = 208.6 ±
4.3; UN = 183.3 ± 4.3), 60 days (C = 308.7 ± 6.6;
UN = 246.1 ± 5.6) and 90 days (C = 378.2 ± 7.7;
UN = 313.1 ± 4.3; Fig. 1).
Between days 4 and 7 in differentiation medium (DM),
both myoblasts from controls and undernourished rats
began to form myotubes into the standardized mode.
Examination of cultures under the phase contrast micro-
scope suggested that on the 10th day of culture, myotubes
from control rats are large and numerous, while the number
and the size of myotubes were low in undernourished rats
(Fig. 2a). LDH measurement showed a lower cell number
in the UN group when compared with cells from the
C group (C group 21.8 ± 0.19 x 103, UN group 18.9 ±
0.2 x 103; Fig. 2b).
Myotubes from UN 90-day-old offspring were less
ramified than those from their controls (Fig. 3).
The expression of Pax7 protein in lysates of cells taken
from 42-, 60- and 90-day-old pups and cultured for 0 and
4 days was studied by western blotting. The expression
remained roughly constant in cells from offspring aged
between 42 and 90 days at all culture times (Fig. 4). There
was no difference in the Pax7 content of cells from UN and
C pups. Similarly, the cadherin (N and M) content of cells
cultured for 4 days was assayed using a pan-cadherin
antibody. The amounts in 42-, 60- and 90-day-old pups
Fig. 1 Body weight of pups from mothers fed a low-protein
diet (undernutrition, casein 8%) or a control diet (casein:17%)
during growth. Analysis was performed every 5 days during lactation
(n = 9 in each group) and at 42, 60 and 90 days (n = 3 in each
group). The values are presented as means + SEM. p < 0.05
compared with control group using two-way ANOVA and
Bonferroni’s post hoc test
appeared to be similar. There was no difference in the
expression of cadherin in cells from UN and C pups
(Fig. 4).
The expression of the b1 integrin subunit in cells cul-
tured for 4 or 7 days did not vary with the age of the pups
providing the muscle samples. The content of the b1
integrin subunit in muscle cultures of UN and C offspring
was similar. Cells from 42-, 60- and 90-day-old pups cul-
tured for 7 days showed similar contents of IL-4Ra, but
IL-4Ra was not detected in cells from 90-day-old off-
spring. The expression of IL-4Ra protein in cells from UN
and C offspring cultured for 7 days was the same.
Muscle cells cultured for 10 days all showed the same
content of myogenin protein, regardless of the age of the
offspring providing the muscle. There was also no differ-
ence in the expression of myogenin in cells cultured for
10 days from 60-day-old UN and C offspring.
The concentration of IL-4 in the supernatants of muscle
cells from 60-day-old offspring cultured for 10 days, nor-
malized using LDH activity, showed no significant differ-
ences between UN and C offspring.
Discussion
Perinatal undernutrition has been one of the most thor-
oughly studied programming factors acting in the genesis
of metabolic disease in adult life [17]. The mechanisms
for the induction of programming appear to include the
following: certain clones of cells may be altered by envi-
ronmental adversity during development; nutrient envi-
ronment may permanently alter gene expression; and early
nutrition may permanently reduce cell numbers in the
organs and tissues [18]. In our study, casein (a highly
phosphorylated protein) was chosen because it is an
important component of breast milk. Casein absorption
induces an increase in the concentrations of essential
(threonine, valine, methionine, isoleucine, leucine, phen-
ylalanine and lysine) and non-essential amino acids (his-
tidine and arginine) in the neonatal plasma [19]. Moreover,
it has been shown that dietary casein intake is the key
factor in the stimulation of ribosome aggregation in liver
that reflects the protein biological value [19]. Thus, casein
has been widely used in previous studies as an inducer of
perinatal undernutrition.
In the present study, we first examined the effects of a
low-protein diet during lactation on the body weight of the
offspring. We then studied the morphology of myotubes
and the expression of some key proteins known to regulate
myogenesis, from muscle-cell cultures of young and adult
offspring. The results indicate that offspring from mothers
fed a low-protein diet showed a lower body weight at adult
age (60 and 90 days) than their controls. Our results are in
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Eur J Nutr
Fig. 2 Morphological
observation of muscle cells in
culture (a) and numbers of
muscle cells/well (0.33 cm2)
(b) after 10 days in culture
(60-day-old offspring). Mothers
were submitted to a control diet
(C1, C2, C3) or a low-protein
diet (UN1, UN2, UN3) during
lactation. a Cells were cultured
in a differentiation medium
(DM) and observed at 10 days.
Phase contrast objective 10x.
Bar 100 µm. b Data were
obtained from LDH activity of
adherent cells. Data are
expressed as mean + SEM
n = 18 in each group.
*p < 0.05 unpaired Student’s
t-test. C control group,
UN undernourished group
agreement with earlier studies using the same experimental
model [11, 20, 21]. Nevertheless, 90-day-old rats were not
evaluated in those studies. Neonatal maternal protein
restriction (8% casein-restricted diet) causes changes in
milk composition and volume of the lactating rats, and it
may be related to less transfer of nutrients to the offspring
and to a long-lasting low body weight [22]. In addition, it
has been demonstrated that reduced body weight induced
by maternal undernutrition is related to the low number of
muscle fibers in offspring [8, 18]. Thus, it can be suggested
that changes in the numbers and/or types of fibers present
in skeletal muscle may contribute to the risk of developing
type 2 diabetes, and/or obesity in later life (developmental
origin of adult disease) [17]. However, little is known
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Eur J Nutr
Fig. 3 Morphological analysis
of muscle cells in culture
(90-day-old offspring).
Mothers’ offspring were
submitted to a control diet
(C1, C2, C3) or a low-protein
diet (UN1, UN2, UN3) during
lactation. Cells were cultured in
a differentiation medium (DM)
and analyzed on the 10th day.
Phase contrast objective 10x.
Bar 100 µm
about the molecular mechanisms by which maternal
nutrition influences muscle growth.
In order to test the hypothesis that neonatal undernu-
trition can impair myogenesis, the capacity of muscles
from offspring of different ages (42, 60 and 90 days) to
produce myogenic markers of satellite cells, myoblasts and
myotubes in culture was assessed. Our data demonstrate
that muscle cells from 60-day-old UN offspring cultured
for 10 days grew more slowly than did muscle cells from C
offspring. There were fewer myotubes in samples from
60-day-old UN offspring, and the myotubes from 90-day-old
offspring expanded more slowly than those from their C
counterparts. It has been demonstrated that muscle-fiber
formation and postnatal growth are regulated by insulin-
like growth factor (IGF)-1 [23]. Systemic IGF-1 levels are
low in both mothers and fetuses following maternal
undernutrition, suggesting that impaired myogenesis could
be the result of diminished levels of IGF-1 [24]. After birth,
IGF-1 has been shown to promote muscle-fiber hypertro-
phy by a combination of protein synthesis and activation of
satellite cells [25]. We therefore performed key protein-
expression analyses of satellite cells, myoblasts and myo-
tubes in the offspring.
Pax7 transcription factor and cadherins (especially N
and M cadherins) are involved in initial cell to cell rec-
ognition during myogenesis and play an important role as
regulators of muscle-cell specification and tissue formation
during development [12, 13]. Pax7 is expressed in quies-
cent satellite cells, serving as a marker for their localization
beneath the basal lamina [12]. M-cadherin accumulates
with b-catenin at the areas of contact between fusing
myoblasts and b-catenin colocalizes with actin in pre-fus-
ing myoblasts [13]. Satellite cells contain b-catenin that
remains present in them during activation and proliferation.
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Eur J Nutr
Fig. 4 Immunoblots of Pax7
and M–N (pan) cadherins in
muscle cells—from 60-day-old
rats. Muscle cells from control
(C1, C2, C3) and
undernourished offspring
(UN1, UN2, UN3) were
cultured for 4 days. The lysates
were separated by 7.5% SDS
PAGE. Actin was used as
control for gel loading. The
amount of each factor relative
to that of actin is shown below.
NS non-significant
In the present study, we found no difference in the
expression of Pax7 and cadherin proteins in cells from UN
and C offspring, regardless of the age of the donor off-
spring or the time in culture. The expression of those
proteins seems to remain roughly the same in offspring
aged between 42 and 90 days. Most satellite cells
expressing Pax7 up-regulate MyoD and enter into a pro-
liferative state [12, 26]. N and M cadherins play a role in
the initiation of myoblast fusion to form multinuclear
myotubes [27]. Our results show that the initial processes
of proliferation and differentiation in myogenesis were not
affected by neonatal undernutrition.
Skeletal muscle cells are known to produce a wide
variety of integrins. The b1 subunit is produced throughout
myogenesis, and different variants are present in myoblasts
and myotubes [28]. Pure populations of b1-null myoblasts
and satellite cells isolated from b1-null chimeric embryos
and chimeric newborn mice differentiate in vitro and fuse
to form multinucleated myotubes [29]. Other studies have
demonstrated that b1-deficient myoblasts adhere to each
other, but that their plasma membrane breakdown is
defective [30]. The amounts of the b1 integrin protein did
not seem to change with the age of the donor pups, and we
found no differences in its expression between cells from
UN and C pups. Many cell adhesion proteins, including the
integrins and cadherins, have been shown to be important
for myoblast fusion, but how exactly they regulate cell
fusion remains largely unknown. However, the focal
adhesion kinase (FAK) involved in integrin signaling
appears to be a mediator by which integrins regulate
myoblast fusion [31]. IL-4 is not required for fusion
between mononucleated myoblasts, but it is required for
myotube maturation. It may favor myogenic cell migration
and the integrin-modulated interaction of these myoblasts
with newly formed myotubes [14].
The newly formed myotubes recruit myoblasts for
fusion by secreting IL-4, leading to muscle growth. The
transcription factor NFATc2 controls myoblast fusion after
the formation of a myotube and is necessary for further cell
growth [14]. IL-4 has been identified as a molecular signal
that binds to the IL-4Ra subunit on myoblasts to promote
fusion and growth [14]. The IL-4Ra subunit of the IL-4R is
present on both myoblasts and myotubes and is required for
muscle growth. IL-4 controls cell fusion by binding to the
IL-4R of myoblasts rather than myotubes, and signaling via
the IL-4R in myoblasts is required for cell fusion with
nascent myotubes [14]. We believe that the IL-4Ra protein
is absent from the muscles of pups aged over 60 days, and
this was true for both UN and C rats. Our results suggest
that this regulation no longer exists in adult rats (60-day-
old). Cells from 60-day-old UN and C rats in culture for
10 days secrete the same amount of IL-4 into the medium.
This suggests that myotube function is not altered in UN
rats, in spite of the difference observed in their expansion.
Lastly, we analyzed the production of the myogenic
regulatory factor myogenin, which is abundant in the
muscles of newborn pups. It resides in the nucleus and may
function as a sequence-specific DNA-binding factor that
interacts directly with muscle-specific genes during myo-
genesis [12]. The content of myogenin protein in 10-day
muscle-cell cultures was not influenced either by the age of
the donor rats or whether they were UN or C rats.
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Eur J Nutr
Conclusion
In conclusion, casein restriction during lactation followed
by the restoration of a normal protein diet at weaning has
no impact on the synthesis of myogenic marker proteins,
and myocyte differentiation occurred normally in the
muscles of offspring aged 42, 60 and 90 days. Neverthe-
less, the number and morphology of myotubes are affected.
Acknowledgments The authors wish to thank CAPES-COFECUB
(grant 584/07) and the National Council for Science and Technology
(CNPq), Brazil, for their financial support.
Conflict of interest The authors declare that they have no conflict
of interest.
References
1. Waterland RA, Jirtle RL (2004) Early nutrition, epigenetic
changes at transposons and imprinted genes, and enhanced sus-
ceptibility to adult chronic diseases. Nutrition 20:63–68
2. Lucas A (2005) Long-term programming effects of early nutri-
tion—implications for the preterm infant. J Perinatol
25(Suppl
2):S2–S6
3. Kind KL, Simonetta G, Clifton PM, Robinson JS, Owens JA
(2002) Effect of maternal feed restriction on blood pressure in the
adult guinea pig. Exp Physiol 87:469–477
4. Ozanne SE, Hales CN (2002) Early programming of glucose-
insulin metabolism. Trends Endocrinol Metab 13:368–373
5. Hales CN, Barker DJ (1992) Type 2 (non-insulin-dependent)
diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia
35:595–601
6. Ravelli GP, Stein ZA, Susser MW (1976) Obesity in young men
after famine exposure in utero and early infancy. N Engl J Med
295:349–353
7. Sawaya AL, Martins PA, Grillo LP, Florencio TT (2004) Long-
term effects of early malnutrition on body weight regulation. Nutr
Rev 62:S127–S133
8. Brameld JM (2004) The influence of undernutrition on
skeletal muscle development. Br J Nutr 91:327–328
9. Mallinson JE, Sculley DV, Craigon J, Plant R, Langley-
Evans SC, Brameld JM (2007) Fetal exposure to a maternal low-
protein diet during mid-gestation results in muscle-specific
effects on fibre type composition in young rats. Br J Nutr
98:292–299
10. Wilson SJ, Ross JJ, Harris AJ (1988) A critical period for
for- mation of secondary myotubes defined by prenatal
undernour- ishment in rats. Development 102:815–821
11. Bayol S, Jones D, Goldspink G, Stickland NC (2004) The
influence of undernutrition during gestation on skeletal muscle
cellularity and on the expression of genes that control
muscle growth. Br J Nutr 91:331–339
12. Perdiguero E, Sousa-Victor P, Ballestar E, Munoz-Canoves
P (2009) Epigenetic regulation of myogenesis.
Epigenetics
4:541–550
13. Wrobel E, Brzoska E, Moraczewski J (2007) M-cadherin
and beta-catenin participate in differentiation of rat satellite cells.
Eur J Cell Biol 86:99–109
14. Horsley V, Jansen KM, Mills ST, Pavlath GK (2003) IL-4 acts as
a myoblast recruitment factor during mammalian muscle growth.
Cell 113:483–494
15. Hirabara SM, Silveira LR, Alberici LC, Leandro CV, Lamb-
ertucci RH, Polimeno GC, Cury Boaventura MF, Procopio J,
Vercesi AE, Curi R (2006) Acute effect of fatty acids on
metabolism and mitochondrial coupling in skeletal muscle. Bio-
chim Biophys Acta 1757:57–66
16. Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72:248–254
17. Barker DJ (2007) The origins of the developmental origins
theory. J Intern Med 261:412–417
18. Sayer AA, Cooper C (2005) Fetal programming of body
com- position and musculoskeletal development. Early Hum
Dev
81:735–744
19. Galibois I, Parent G, Savoie L (1987) Effect of dietary proteins
on time-dependent changes in plasma amino acid levels and on
liver protein synthesis in rats. J Nutr 117:2027–2035
20. Pires-de-Melo IH, Wanderley Dos Reis F, Luz LS, Paz ST, Silva
HJ, Souza SL, Leandro CG (2009) Short- and long-term effects of
a neonatal low-protein diet in rats on the morphology of the
larynx. Nutrition 25:855–860
21. Wilson MR, Hughes SJ (1997) The effect of maternal protein
deficiency during pregnancy and lactation on glucose tolerance
and pancreatic islet function in adult rat offspring. J Endocrinol
154:177–185
22. Passos MC, da Fonte Ramos C, Dutra SC, Mouco T, de Moura
EG (2002) Long-term effects of malnutrition during lactation on
the thyroid function of offspring. Horm Metab Res 34:40–43
23. Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-
Meek S, Dalton D, Gillett N, Stewart TA (1993) IGF-I is required
for normal embryonic growth in mice. Genes Dev 7:2609–2617
24. Dwyer CM, Stickland NC, Fletcher JM (1994) The influence of
maternal nutrition on muscle fiber number development in the
porcine fetus and on subsequent postnatal growth. J Anim Sci
72:911–917
25. Barton-Davis ER, Shoturma DI, Sweeney HL (1999) Contribu-
tion of satellite cells to IGF-I induced hypertrophy of
skeletal muscle. Acta Physiol Scand 167:301–305
26. Zammit PS, Relaix F, Nagata Y, Ruiz AP, Collins CA, Partridge
TA, Beauchamp JR (2006) Pax7 and myogenic progression in
skeletal muscle satellite cells. J Cell Sci 119:1824–1832
27. Hollnagel A, Grund C, Franke WW, Arnold HH (2002) The cell
adhesion molecule M-cadherin is not essential for muscle
development and regeneration. Mol Cell Biol 22:4760–4770
28. Bozyczko D, Decker C, Muschler J, Horwitz AF (1989) Integrin
on developing and adult skeletal muscle. Exp Cell Res 183:72–91
29. Crawley S, Farrell EM, Wang W, Gu M, Huang HY, Huynh V,
Hodges BL, Cooper DN, Kaufman SJ (1997) The alpha7beta1
integrin mediates adhesion and migration of skeletal myoblasts
on laminin. Exp Cell Res 235:274–286
30. Schwander M, Leu M, Stumm M, Dorchies OM, Ruegg
UT, Schittny J, Muller U (2003) Beta1 integrins regulate
myoblast fusion and sarcomere assembly. Dev Cell 4:673–685
31. Quach NL, Biressi S, Reichardt LF, Keller C, Rando TA (2009)
Focal adhesion kinase signaling regulates the expression of
caveolin 3 and beta1 integrin, genes essential for normal
myo- blast fusion. Mol Biol Cell 20:3422–3435
77
78
7. CONCLUSÕES
Os resultados desse estudo permitem concluir que:
A desnutrição neonatal, mesmo seguida de reposição nutricional, modifica a
quantidade de macrófagos alveolares em cultura de ratos jovens e adultos, o que pode
interferir no desenvolvimento dessas células em cultura. Além disso, altera a produção
de proteínas de fusão (IL-4 e IFNγ), sem modificar a expressão de caderinas na
superfície dos macrófagos. Apesar dessas alterações observadas, devido a um provável
mecanismo compensatório existente nos animais desnutridos, pode-se sugerir que a
formação das células gigantes multinucleares mediada por caderinas e IFNγ parece
não ser afetada na vida adulta do animal pela restrição protéica na lactação.
A desnutrição neonatal, mesmo seguida de reposição nutricional, reduz a quantidade
de miotubos em cultura de ratos com 60 dias de vida e altera a morfologia dessas
células em ratos adultos com 90 dias de vida, os quais apresentam miotubos com
menos ramificações. Dessa forma, pode-se sugerir que esse modelo de desnutrição
compromete o desenvolvimento da miogênese. No entanto, a expressão de proteínas-
chaves do processo miogênico permanece inalterada.
79
8. RECOMENDAÇÕES/PERSPECTIVAS
Perspectivas elaboradas como objetivo para dar continuidade ao estudo:
Avaliar as repercussões tardias da desnutrição numa fase mais precoce do
desenvolvimento, o período gestacional, sobre a fusão de macrófagos e células
musculares esqueléticas.
Estudar o efeito da desnutrição neonatal sobre a contração de miotubos em cultura e a
ação da insulina na formação dessas células in vitro (tese de doutorado da aluna
Simone do Nascimento Fraga).
Utilizar o aleitamento cruzado para estudo das repercussões tardias da desnutrição
neonatal.
Avaliar a influência da atividade física sobre a expressão de proteínas de fusão
macrofágica e miocitária em ratos submetidos à desnutrição precoce.
Realizar co-cultura de macrófagos alveolares com miotubos recém formados e
verificar o efeito da desnutrição precoce no índice de fusão dessas células in vitro.
80
9. REFERÊNCIAS
ALLENDE, L.M. et al. Immunodeficiency associated with anorexia nervosa is secondary and
improves after refeeding. Immunology, v.94 (4), p.543-551, 1998.
AMERSFOORT, E.S.V.; BERKEL, T.J.C.V.; KUIPER, J. Receptors, Mediators, and
Mechanisms Involved in Bacterial Sepsis and Septic Shock. Clin. Microbiol. Rev., v.16 (3),
p.379-414, 2003.
ANGST, B.D.; MARCOZZI, C.; MAGEE, A.I. The cadherin superfamily: diversity in form
and function. J. Cell Sci., v.114, p.629-641, 2001.
ARABABADI, M.K. et al. Evaluation of relation between IL-4 and IFN-γ polymorphisms and
type 2 diabetes. Iran J. Basic Med. Sci., v.12, p.100-104, 2009.
ARAÚJO, F.R.G. et al. Perialveolar bacterial microbiota and bacteraemia after dental
alveolitis in adult rats that had been subjected to neonatal malnutrition. Br. J. Nutr.
(Epublication ahead of print version), 2011.
BARKER, D.J. Early growth and cardiovascular disease. Arch. Dis. Child, v.80, p.305-307,
1999.
BARKER, D.J. The developmental origins of adult disease. Eur. J. Epidemiol., v.18, p.733-
736, 2003.
BARKER, D.J. The origins of the developmental origins theory. J. Intern. Med., v.261 (5),
p.412-417, 2007.
BARRETO-MEDEIROS, J.M. et al. Malnutrition during brain growth spurt alters the effect
of fluoxetine on aggressive behavior in adult rats. Nutr. Neurosci., v.7, p.49-52, 2004.
BARROS, K.M.F.T. et al. A regional model (Northeastern Brazil) of induced mal-nutrition
delays ontogeny of reflexes and locomotor activity in rats. Nutr. Neurosci., v.9, p.99-104,
2006.
BARTON-DAVIS, E.R.; SHOTURMA, D.I.; SWEENEY, H.L. Contribution of satellite cells
to IGF-I induced hypertrophy of skeletal muscle. Acta Physiol. Scand., v.167 (4), p.301-305,
1999.
BATISTA FILHO, M.; RISSIN, A. A transição nutricional no Brasil: tendências regionais e
temporais. Cad. Saúde Pública, Rio de Janeiro, v.19 (Sup. 1), p.S181-S191, 2003.
BAYOL, S. et al. The influence of undernutrition during gestation on skeletal muscle
cellularity and on the expression of genes that control muscle growth. Br. J. Nutr., v.91,
p.331-339, 2004.
81
BELPERIO, J.A. et al. The role of cytokines during the pathogenesis of ventilator-associated
and ventilatorinduced lung injury. Semin. Respir. Crit. Care Med., v.27, p.350-364, 2006.
BORBA, J.M.C. et al. Permanent and transitory morphometric changes of NADPH-
diaphorase-containing neurons in the rat visual cortex after early malnutrition. Brain Res.
Bull., v.53 (2), p.193-201, 2000.
BORELLI, P. et al. Protein-energy malnutrition halts hemopoietic progenitor cells in the
G0/G1 cell cycle stage, thereby altering cell production rates. Braz. J. Med. Biol. Res., v.42,
p.523-530, 2009.
BOZYCZKO, D. et al. Integrin on developing and adult skeletal muscle. Exp. Cell Res., v.183
(1), p.72-91, 1989.
BRADFORD, M.M. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem., v.72,
p.248-254, 1976.
BRAMELD, J.M. The influence of undernutrition on skeletal muscle development. Br. J.
Nutr., v. 91 (3), p.327-328, 2004.
BUCK, M.; CHOJKIER, M. Muscle wasting and dedifferentiation induced by oxidative stress
in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and
antioxidants. EMBO J., v.15, p.1753–1765, 1996.
BURET, A.G. Pathophysiology of enteric infections with Giardia duodenalius. Parasite., v.15
(3), p.261-265, 2008.
BUTERA WADSWORTH, D.A. Characterization of disintegrins of Viperidae venoms as
selective tools in the detection or inhibition of the function of integrins. 2003. Tese
(Doutorado) - Instituto de Química, Universidade de São Paulo, São Paulo, 2003.
CAVALCANTE, R.B. Alterações nos genes da E-caderina e β-catenina em adenoma
pleomórfico e carcinoma adenóide cístico: estudo molecular e imuno-histoquímico. 2008.
Tese (Doutorado) - Universidade Federal do Rio Grande do Norte, Natal, 2008.
CHAN, J. et al. Effects of protein calorie malnutrition on mice infected with BCG. J. Nutr.
Immunol., v.5, p.11-19, 1997.
CHANDRA, R.K. Nutrition and the immune system: an introduction. Am. J. Clin. Nutr., v.66
(2), p.460-463, 1997.
CHANDRA, R.K. Nutrition and the immune system from birth to old age. Eur. J. Clin. Nutr.,
v.3, p.73-76, 2002.
CHARGÉ, S.B.P. Interleukine-4 et fusion des cellules musculaires. M/S., v.19 (12), p.1185-
1187, 2003.
82
CHARGÉ, S.B.P.; RUDNICKI, M.A. Cellular and molecular regulation of muscle
regeneration. Physiol. Rev., v.84, p.209–238, 2004.
CONRAADS, V. Pro-inflammatory cytokines and their receptors in chronic heart failure: do
they really matter? Acta Cardiol., v.61, p.161–168, 2006.
COSSU, G. et al. Activation of different myogenic pathways: myf-5 is induced by the neural
tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm. Development., v.122,
p.429-437, 1996.
CRAWLEY, S. et al. The alpha7 beta1 integrin mediates adhesion and migration of skeletal
myoblasts on laminin. Exp. Cell Res., v.235 (1), p.274-286, 1997.
D'ACAMPORA, A.J. et al. Pulmão e Peritônio após Sepse Experimental de Origem
Abdominal em Ratos Wistar. Revista do Colégio Brasileiro de Cirurgiões, v.28 (1), p.276,
2001.
DÂMASO, A. Nutrição e exercício na prevenção de doenças. Rio de Janeiro: MEDSI, 2001.
DE CASTRO, C.M.M.B. et al. Modulation by dexamethasone of phospholipase A2 activities
in endotoxemic guinea pigs. J. Appl. Physiol., v.79, p.1271-1277, 1995.
DE CASTRO, C.M.M.B. et al. LPS bacteriano: um mediador de inflamação. An. Fac. Med.
Univ. Fed. Pernamb., v.42 (2), p.78-83, 1997.
DWYER, C.M.; STICKLAND, N.C.; FLETCHER, J.M. The influence of maternal nutrition
on muscle fiber number development in the porcine fetus and on subsequent postnatal growth.
J. Anim. Sci., v.72 (4), p.911-917, 1994.
FAO. The State of Food and Agriculture 2010-11. Food and Agriculture Organization of the
United Nations, Roma, 2011. Disponível em:<http://www.fao.org/publications/sofa/en/>.
Acesso em: 13 jun. 2011.
FEFERBAUN, R. et al. Gasto energético no recém-nascido e lactente com sepse. Rev. Bras.
Crescimento Desenvolv. Hum., v.19 (1), p.122-133, 2009.
FERNANDEZ-BOYANAPALLI, F. et al. Impaired apoptotic cell clearance in CGD due to
altered macrophage programming is reversed by phosphatidylserine-dependent production of
IL-4. Blood, v. 113, p.2047-2055, 2009.
FERREIRA E SILVA, W.T. et al. Perinatal Malnutrition Programs Sustained Alterations in
Nitric Oxide Released by Activated Macrophages in Response to Fluoxetine in Adult Rats.
Neuroimmunomodulation., v.16, p.219-227, 2009.
83
FOCK, R.A. et al. Protein-energy malnutrition modifies the production of interleukin-10 in
response to lipopolysaccharide (LPS) in a murine model. J. Nutr. Sci. Vitaminol., v.54, p.371-
377, 2008.
FOSCHINI, R.M.S.A.; RAMALHO, F.S.; BICAS, H.E.A. Myogenic satellite cells. Arq. Bras.
Oftalmol., v.67 (4), p.681-687, 2004.
FRICK, C.G. et al. Chronic Escherichia coli infection induces muscle wasting without
changing acetylcholine receptor numbers. Intensive Care Med., v.34, p.561-567, 2008.
GOLDBERG, A.L. et al. Activation of protein breakdown and prostaglandin E2 production in
rat skeletal muscle in fever is signaled by a macrophage product distinct from interleukin 1 or
other known monokines. J. Clin. Invest., v.81, p.1378-1383, 1988.
GUBERNATIS, H. Progress for Children: a Report Card on Nutrition. UNICEF, 2006.
HALES, C.N.; BARKER, D.J. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty
phenotype hypothesis. Diabetologia, v.35 (7), p.595-601, 1992.
HALEVY, O. et al. Early posthatch feeding stimulates satellite cell proliferation and skeletal
muscle growth in turkey poults. J. Nutr., v.133, p.1376–1382, 2003.
HASSELGREN, P.O; FISCHER, J.E. Sepsis: Stimulation of Energy-Dependent Protein
Breakdown Resulting in Protein Loss in Skeletal Muscle. World J. Surg., v.22, p.203-208,
1998.
HELMING, L.; GORDON, S. The molecular basis of macrophage fusion. Immunobiol, v.212,
p.785-793, 2008.
HELMING, L.; GORDON, S. Molecular mediators of macrophage fusion. Trends in Cell
Biology, v.19, p.514-522, 2009.
HIRABARA, S.M. et al. Acute effect of fatty acids on metabolism and mitochondrial
coupling in skeletal muscle. Biochim. Biophys. Acta, v.1757 (1), p.57-66, 2006.
HOLLNAGEL, A. et al. The cell adhesion molecule M-cadherin is not essential for muscle
development and regeneration. Mol. Cell Biol., v.22 (13), p.4760-4770, 2002.
HORSLEY, V. et al. IL-4 acts as a myoblast recruitment factor during mammalian muscle
growth. Cell., v.113, p.483-494, 2003.
ISOBE, A; HIRAYAMA, E; KIM, J. Characterization of myogenin expression in myotubes
derived from quail myoblasts transformed with a temperature sensitive mutant of Rous
sarcoma vírus. Cell Struct. Funct., v.23, p.57-67, 1998.
JEPSON, M. et al. Effects of endotoxemia on protein metabolism in skeletal muscle and liver
of fed and fasted rats. Biochem. J., v.235, p.329-336, 1986.
84
KIND, K.L. et al. Effect of maternal feed restriction on blood pressure in the adult guinea pig.
Exp. Physiol., v.87 (4), p.469-477, 2002.
KLEIN, D.J. et al. Endotoxemia related to cardiopulmonary bypass is associated with
increased risk of infection after cardiac surgery: a prospective observational study. Critical
Care, v.15:R69, 2011.
KRISTINA, N.; HERBERT, L; MATTHIAS, P. Muscle dysfunction in malnutrition. Curr.
Nutr. Food Sci., v.1 (3), p.253-258, 2005.
LANDGRAF, M.A.V. et al. Intrauterine undernutrition in rats interferes with leukocyte
migration, decreasing adhesion molecule expression in leukocytes and endothelial cells. J.
Nutr., v.135, p.1480-1485, 2005.
LI, Y.P. et al. TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-
regulating UbcH2/E220k. FASEB J., v.17, p.1048-1057, 2003.
LUCAS, A. Programming by early nutrition in man. Ciba Found Symp., v.156, p.38-50, 1991.
LUCAS, A. Long-term programming effects of early nutrition -- implications for the preterm
infant. J Perinatol., v.25 (Sup. 2), p.S2-S6, 2005.
MACLAUCHLAN, S. et al. Macrophage fusion, giant cell formation, and the foreign body
response require matrix metalloproteinase 9. J. Leukoc. Biol., v.85, p.617-626, 2009.
MALLINSON, J.E. et al. Fetal exposure to a maternal low-protein diet during mid-gestation
results in muscle-specific effects on fibre type composition in young rats. Br. J. Nutr., v.98
(2), p.292-299, 2007.
MARTINEZ, F.O.; HELMING, L.; GORDON, S. Alternative activation of macrophages: an
immunologic functional perspective, Annu. Rev. Immunol., v.27, p. 451–483, 2009.
MÁRTON, I.J.; KISS, C. Protective and destructive immune reactions in a apical
periodontitis. Oral Microbiol. Imunol., v.15, p.139-150, 2000.
McINNES, A.; RENNICK, D.M. Interleukin 4 induces cultured monocytes/macrophages to
form giant multinucleated cells. J. Exp. Med., v.167, p.598–611, 1988.
McNALLY, A. K.; ANDERSON, J. M. Interleukin-4 induces foreign body giant cells from
human monocytes/macrophages. Differential lymphokine regulation of macrophage fusion
leads to morphological variants of multinucleated giant cells. Am. J. Pathol., v.147, p.1487-
1499, 1995.
McNALLY, A.K.; ANDERSON, J.M. β1 and β2 integrins mediate adhesion during
macrophage fusion and multinucleated foreign body giant formation. Am. J. Pathol., v. 160,
p.621-630, 2002.
85
MEGE, R.M. et al. N-cadherin and N-CAM in myoblast fusion: compared localisation and
effect of blockade by peptides and antibodies. J. Cell Sci., v.103, p.897-906, 1992.
MELO, J.F. et al. Effect of neonatal malnutrition on cell recruitment and oxidant-antioxidant
activity of macrophages in endotoxemic adult rats. Rev. Nutr., v.21, p. 683-694, 2008.
MELO, J.F. et al. Effect of a neonatal low-protein diet on the morphology of myotubes in
culture and the expression of key proteins that regulate myogenesis in young and adult rats.
Eur. J. Nutr., v.50, p.243-250, 2011.
MENG, F.; LOWELL, C.A. Lipopolysaccharide (LPS)-induced macrophage activation and
signal transduction in the absence of Src-Family Kinases Hck, Fgr, and Lyn. J.Exp.Med.,
v.185 (9), p.1661-1670, 1997.
MEYER, J. et al. Increased organ blood flow in chronic endotoxemia is reversed by nitric
oxide synthase inhibition. J. Appl. Physiol., v.76, p.2785-2793, 1994.
MORENO, J.L. et al. IL-4 promotes the formation of multinucleated giant cells from
macrophage precursors by a STAT6-dependent, homotypic mechanism: contribution of E-
cadherin. J. Leukoc. Biol., v.82, p.1542–1553, 2007.
MUSCARITOLI, M. et al. Prevention and treatment of cancer cachexia: new insights into an
old problem. Eur. J. Cancer, v.42, p.31-41, 2006.
OZANNE, S.E.; HALES, C.N. Early programming of glucose-insulin metabolism. Trends
Endocrinol Metab., v.13 (9), p.368-373, 2002.
PASSOS, M.C.; RAMOS, C.F.; MOURA, E.G. Short and long term effects of malnutrition in
rats during lactation on the body weight of offsprings. Nutr. Res., v.20, p.1603-1612, 2000.
PASSOS, M.C. et al. Long-term effects of malnutrition during lactation on the thyroid
function of offspring. Horm. Metab. Res., v.34 (1), p.40-43, 2002.
PAVLATH, G.K.; HORSLEY, V. Cell fusion in skeletal muscle--central role of NFATC2 in
regulating muscle cell size. Cell Cycle, v.2, p.420-423, 2003.
PERDIGUERO, E. et al. Epigenetic regulation of myogenesis. Epigenetics, v.4 (8), p.541-
550, 2009.
PIRES-DE-MELO, I.H. et al. Short- and long-term effects of a neonatal low-protein diet in
rats on the morphology of the larynx. Nutrition, v.25 (7-8), p.855-860, 2009.
POWELL-BRAXTON, L. et al. IGF-I is required for normal embryonic growth in mice.
Genes Dev 7(12B):2609-2617, 1993.
86
QUACH, N.L. et al. Focal adhesion kinase signaling regulates the expression of caveolin 3
and beta1 integrin, genes essential for normal myoblast fusion. Mol. Biol. Cell., v.20 (14),
p.3422-3435, 2009.
RAVELLI, G.P.; STEIN, Z.A.; SUSSER, M.W. Obesity in young men after famine exposure
in utero and early infancy. N. Engl. J. Med., v.295 (7), p.349-353, 1976.
REEVES, P.G.; IELSEN, F.H.; FAHEY, G.C. AIN-93 purified diets for laboratory rodents:
final report of the American Institute of Nutrition Ad Hoc writing Committee on the
reformulation of the AIN-76 A Rodent diet. J. Nutr., v.123, p.1939-1951, 1993.
RODRÍGUEZ, L. et al. Assessment by flow cytometry of cytokine production in
malnourished children. Clin. Diagn. Lab. Immunol., v.12, p.502-507, 2005.
RUFF, R.; SECRIST, D. Inhibitors of prostaglandin synthesis or cathepsin D prevent muscle
wasting due to sepsis in the rat. J. Clin. Invest., v.73, p.1483-1486, 1984.
RUFINO, R.; SILVA, J.R.L. Bases celulares e bioquímicas da doença pulmonar obstrutiva
crônica. J.Bras.Pneumol., v.32 (3), p.241-248, 2006.
SAWAYA, A.L. et al. Obesity and malnutrition in a Shantytown population in the city of Sao
Paulo, Brazil. Obes. Res., v.3, p.S107-115, 1995.
SAWAYA, A.L. et al. Long-term effects of early malnutrition on body weight regulation.
Nutrition reviews, v.62 (7 Pt 2), p.S127-133, 2004.
SAYER, A.A.; COOPER, C. Fetal programming of body composition and musculoskeletal
development. Early Hum. Dev., v.81 (9), p.735-744, 2005.
SCHWANDER, M. et al. Beta1 integrins regulate myoblast fusion and sarcomere assembly.
Dev. Cell, v.4, p.673-685, 2003.
SERAFIM, T.D. et al. Immune response to Leishmania (Leishmania) chagasi infection is
reduced in malnourished BALB/c mice. Mem. Inst. Oswaldo Cruz, v.105, p.811-817, 2010.
SUNIL, V.R. et al. Activation of adherent vascular neutrophils in the lung during acute
endotoxemia. Respir. Res., v.3 (1), p.3-21, 2002.
SUPINSKI, G.S; CALLAHAN, L.A. Free radical-mediated skeletal muscle dysfunction in
inflammatory conditions. J. Appl. Physiol., v.102, p.2056-2063, 2007.
TE PAS, M.F. et al. Influences of myogenin genotypes on birth weight, growth rate, carcass
weight, backfat thickness, and lean weight of pigs. J. Anim. Sci., v.77, p.2352-2356, 1999.
VIGNERY, A. Macrophage fusion: the making of osteoclasts and giant cells. JEM, v. 202,
p.337-340, 2005.
87
VILLARROEL, M.V. et al. Citocinas TH2 (IL4 e IL10) en el niño desnutrido. Arch. Venez.
Puer. Ped., v.71, p.42-47, 2008.
WANG, C.Z. et al. Pulmonary inflammatory cell response to sustained endotoxin
administration. J. Appl. Physiol., v.76 (2), p.516-522, 1994.
WATERLAND, R.A.; JIRTLE, R.L. Early nutrition, epigenetic changes at transposons and
imprinted genes, and enhanced susceptibility to adult chronic diseases. Nutrition, v.20 (1),
p.63-68, 2004.
WEINMAN, A.R. et al. Assessment of vitamin A nutritional status in newborn preterm
infants. Nutrition, v.23, p.454-460, 2007.
WEINSTEIN, S.L. et al. Phosphatidylinositol 3-kinase and mTOR mediate
lipopolysaccharide-stimulated nitric oxide production in macrophages via interferon-b. J.
Leukoc. Biol., v.67, p.405-414, 2000.
WHEELOCK, M.J.; JOHNSON, K.R. Cadherins as modulators of cellular phenotype. Annu
Rev. Cell Dev. Biol., v.19, p.207-235, 2003.
WILSON, S.J.; ROSS, J.J.; HARRIS, A.J. A critical period for formation of secondary
myotubes defined by prenatal undernourishment in rats. Development, v.102, p.815-821,
1988.
WRÓBEL, E.; BRZÓSKA, E.; MORACZEWSKI, J. M-cadherin and beta-catenin participate
in differentiation of rat satellite cells. Eur. J. Cell Biol., v.86, p.99-109, 2007.
YOUNG, H.A.; HARDY, K.J. Role of interferon-γ in immune cell regulation. J. Leukoc.
Biol., v.58, p.373-381, 1995.
ZAMMIT, P.S. et al. Pax7 and myogenic progression in skeletal muscle satellite cells. J. Cell
Sci., v.119 (Pt 9), p.1824-1832, 2006.
88
ANEXOS
Anexo A. Aprovação do Comitê de Ética em Experimentação Animal da UFPE
89
Anexo B. Documentação do Artigo 1
Enviada: sexta-feira, 3 de fevereiro de 2012 00:34:05
Para: [email protected]
Dear Mrs. MELO,
On 2nd Feb 2012, we received your manuscript entitled "Long-term effects of a neonatal low-
protein diet in rats on the number of macrophages in culture and the expression/production of
fusion proteins" by Juliana Melo, Thacianna Costa, Tamara Lima, Maria Chaves, Muriel
Vayssade, Marie-Danielle Nagel, and Célia Castro.
The manuscript has been assigned the Paper number: BJN-2012-018000.
If we have any queries regarding your submission we will contact you within the next few
days.
Sincerely,
Claire Goodstein
Publications Office
British Journal of Nutrition
The Nutrition Society, 10 Cambridge Court, 210 Shepherds Bush Road, London W6 7NJ, UK
Tel: +44 (0)20 7371 6225
Fax: +44 (0)20 7602 1756
E-mail: [email protected]
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Anexo C. Documentação do Artigo 2
Date: 30-08-2010
To: "Juliana Melo" [email protected]
From: "Gerhard Rechkemmer" [email protected]
Subject:
EJN: Your manuscript entitled Effect of a neonatal low-protein diet on the
morphology of myotubes in culture and the expression of key proteins that regulate
myogenesis in young and adult rats
Ref.: Ms. No. EJN-D-10-00050R1
Effect of a neonatal low-protein diet on the morphology of myotubes in culture and the
expression of key proteins that regulate myogenesis in young and adult rats
European Journal of Nutrition
Dear Mrs Melo,
I am pleased to tell you that your work has now been accepted for publication in European
Journal of Nutrition.
It was accepted on 30-08-2010.
Thank you for submitting your work to this journal.
With kind regards
Gerhard Rechkemmer
Editor-in-Chief
European Journal of Nutrition
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Resumos da tese publicados em congressos:
Effect of a neonatal low-protein diet on the cell number and morphology of myotubes
in adult rats In: XV Meeting of the Brazilian Society for Cell Biology, 2010, São
Paulo-SP.
Expressão de Pax7 e miogenina por células satélites musculares e miotubos de ratos
jovens e adultos submetidos à desnutrição neonatal In: V Congresso Brasileiro de
Células-Tronco e Terapia Celular, 2010, Gramado-RS.
Cell number quantification and IL-4 production of macrophages in young and adult
rats submitted to neonatal malnutrition In: XXVI Reunião Anual da Federação de
Sociedades de Biologia Experimental –FESBE, 2011, Rio de Janeiro-RJ.
IL-4Rα/IL-4 in myoblasts and myotubes of young and adult rats submitted to neonatal
malnutrition In: VI Congresso Brasileiro de Células-Tronco e Terapia Celular, 2011,
Salvador-BA.
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